KR20130026976A - Composition for improving obesity and fatty liver using an extract of leaves of sasa quelpaertensis or p-coumaric acid - Google Patents

Abstract

PURPOSE: A composition for treating obesity and fatty liver using a Sasa quelpaertensis leaf effects or para-coumaric acid isolated from the same is provided to enhance an adiponectin expression level and to activate AMPK(AMP-activated protein kinase). CONSTITUTION: A composition for treating obesity contains a Sasa quelpaertensis leaf effects or para-coumaric acid isolated from the same. The extract is prepared using water, ethanol, or a mixture thereof. The composition is a food composition or a pharmaceutical composition.

Description

Composition for improving obesity and fatty liver using an extract of leaves of Sasa quelpaertensis or p-coumaric acid}

The present invention jeju jeori ( Sasa quelpaertensis ) leaf extract or para-coumaric acid (p-coumaric acid) separated from the above relates to a composition for improving obesity and fatty liver.

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. In Korea, the proportion of BMI (25) population was 11.7% in 1995, which is 30.7% in 2008, and the proportion of highly obese (BMI> 30) population is rapidly increasing from 2.3% in 1998 to 4.1% in 2008 (Ministry of Health and Welfare). , 2009).

Obesity is an abnormal increase in fat tissue due to excessive accumulation of energy in the body due to an imbalance between energy intake and consumption (Clinical Endocrinology 28: 675-689, 1998; Clinical Obesity (eds. Kopelman PG, Stock MJ). ) 248-89 (Blackwell Science, Oxford 1998).

The World Health Organization (WHO) estimates that the Asian Standard Body Mass Index (BMI) is 23-25 overweight, 25-30 overweight, and 30 overweight.

The causes of obesity include environmental and genetic factors such as high fat and high calorie diet, lack of exercise due to busy social environment, and endocrine disorders. Among them, about 50 to 70% of obesity is caused by environmental factors. It is known that the rest is due to 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-protein diet, high fat diet, hunger, vitamin deficiency, excessive sugar intake), drug abuse (Sung Hoon Kim et al., "Cause of fatty liver diagnosed by abdominal ultrasound") Korean Journal of Medicine 175 (1995.12) pp. 785-794).

Currently, the most popular treatments for obesity are Orlistat's main ingredients such as Xenical ™ (Roche) and Sibutramine based Reductyl ™ (Abbott), but there are nausea, diarrhea, abdominal pain, insomnia, increased blood pressure and fatty stools. As a therapeutic agent for fatty liver, fibrate-based drugs such as clofibrate are used in clinical practice, but side effects such as liver dysfunction are reported (Atherosclerosis. 92: 31-40 ( 1992).

As the side effects of synthetic medicines and Western medicine are showing limitations, the value of natural medicines is newly emerging.

The present invention discloses the obesity improving activity and fatty liver improvement activity of jeju jeoldae extract.

An object of the present invention is to provide an obesity improver composition and a fatty liver improver composition.

Other objects of the present invention or specific objects will be presented below.

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

As confirmed in the following Examples and Experimental Examples, the present inventors compared with the case of administering Jeju choridae leaf hydrothermal extract with high fat diet alone, the weight gain was significantly reduced and It was confirmed that the degree of increase in abdominal fat and kidney fat was reduced.

In addition, the present inventors in the administration group of jeju jeoldae extract increased the expression level of adiponectin (adiponectin), activates AMP- (AMP-activated protein kinase) (p-AMPK increase) and inhibits the activity of acetyl-CoA carboxylase (ACC) (p -Increased ACC), and cell experiments also confirmed that the jejudae extract activates AMPK and activates CPT-1α (carnitine palmitoyl transferase-1α) (increased CPT-1α).

In addition, the present inventors also found that p -coumaric acid, a bioactive substance isolated from Jeju jeju extract, inhibited the accumulation of triglycerides in cell experiments, increased AMPK phosphorylation and ACC phosphorylation, and activated AMPK. To increase the phosphorylation of LKB1 and to increase the expression of PPARα, a transcription factor of CPT-1.

Increased expression of adiponectin is known to be closely associated with obesity improvement (Commb, TP, et al, J. Clin. Invest., 108 (12): 1875-1881, 2001), and AMPK has been shown to maintain energy homeostasis in cells. As an enzyme that plays a role as a sensor, it is known to be activated when the AMP: ATP ratio is increased by oxidative stress such as glucose deficiency or ROS (J. Biol. Chem. 277: 32571-32577, 2002; J. Appl. Physiol 92: 2475-2482, 2002). When AMPK is activated, ACC is phosphorylated and inactivated. ACC is an enzyme that is involved in the initial stage of fatty acid synthesis and is an enzyme that synthesizes acetyl-CoA into 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, thereby activating UCP2, CPT-1α and the like to promote fat 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 AMPK is activated, the accumulation of fat in the body can be suppressed, and as a result, the increase in body weight can be suppressed, and the result of lowering the concentration of triglycerides and cholesterol in the blood can be obtained.

Obesity improver composition of the present invention is provided based on the results of the above-described experiment, the obesity improver composition of the present invention is characterized in that it comprises jeju jeoldae extract or p -coumaric acid as an active ingredient.

In the present specification, "Jeju jeoldae leaf extract" is used as the extraction target jeju jeoldae leaves, as the extraction solvent, lower alcohols having 1 to 4 carbon atoms, such as water, methanol, ethanol, butanol, ethylene, acetone, hexane, ether, chloroform Means an extract obtained by using ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixed solvent thereof. do. The extraction method may be applied to any method such as cooling, reflux, heating, ultrasonic radiation, taking into account the degree of extraction of the active ingredient, the degree of preservation. In addition, a polysaccharide degrading enzyme may be used at the time of extracting an active substance (an unknown single substance showing activity) or diversifying the active substance, wherein a monosaccharide is formed by binding at least 3 molecules, preferably at least 10 molecules. Any enzyme may be used as long as it can degrade saccharides. Such enzymes include, for example, cellulase, xylanase, hemicellulase and beta-glucanase, cellobihtydrolase, 1,4-beta -Di-glucosidase (1,4-β-D-glucosidase) and 1,4-beta-di-glucanase (1,4-β-D-glucanase), amyloglucosidase, Alpha-amylase, pectinase, pectinase, pentosanase, mannase, pectintranseliminase, polygalactonase, pectinesterase And / or animal or plant extracts containing these enzymes. In practice, Pectinex ™ (Novo Nordisk, Denmark), Ultraflo L ™ (Novo Nordisk, Denmark), Viscozyme L ™ (Novo Nordisk, Denmark), Ceremix ™ (Novo Nordisk, Denmark), Celluclast ™ (Novo Nordisk, Denmark), 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 solid extract from which the extraction solvent is removed in a manner such as 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 addition, the term "active ingredient" as used herein means a component that can exhibit the desired activity alone or in combination with a carrier that is not active itself.

In the present specification, "obesity" means a state in which fat tissue is abnormally increased, whether it is caused by genetic factors or obesity due to environmental factors, and obesity according to the division of body mass index (BMI is 25 or more). ) And overweight (when BMI is 23-25).

In addition, in the present specification, "improving obesity" is meant to include the prevention of obesity, the treatment of obesity, including the reduction of body fat and / or weight loss.

The obesity improver composition of the present invention may include any effective amount (effective amount) according to the use, formulation, formulation purpose, etc., as long as the effective ingredient can exhibit the activity to improve obesity, the usual effective amount is based on the total weight of the composition When determined to be in the range of 0.001% to 99.990% by weight. Here, the "effective amount" refers to the amount of the active ingredient that can induce the effect of improving obesity. Such effective amounts can be determined experimentally within the ordinary skill of those skilled in the art.

Subjects to which the compositions of the invention can be applied (prescribed) are mammals and humans, in particular humans.

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

In another aspect, the present invention relates to a hyperlipidemic improver composition comprising jeju jeoldae extract or p -coumaric acid as an active ingredient.

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

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

In the diet composition and hyperlipidemic composition of the present invention, in relation to the meaning of Jeju jerk extract, the effective amount thereof, and the like described above with respect to the obesity improver composition of the present invention is effective as it is.

In another aspect, the present invention relates to a disease prevention composition caused by the cause of obesity comprising jeju jeoldae extract as an active ingredient.

If obesity persists, obesity may cause hypertension, elevated blood cholesterol, kidney disease, stroke, arteriosclerosis, fatty liver, arthritis, cancer, sleep apnea, and diabetes.

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

Even in the composition for preventing diseases caused by obesity of the present invention, the above-described meanings related to the obesity improver composition of the present invention are effective as it is in relation to the meaning of the jeju jeoldae extract, its effective amount and the like as its active ingredient.

In another aspect, the present invention relates to a fatty liver improver composition comprising jeju jeoldae extract or p -coumaric acid as an active ingredient.

Experimental example below shows that the extracts of Jeju jejudae were the content of glutamate pyruvate transaminase (hereinafter referred to as "GPT"), glutamate oxaloacetate transaminase (hereinafter referred to as "GOT") and lactate dehydrogenase (hereinafter referred to as "LDH"). It was also shown that the liver tissue was similar to that of the normal diet group, but the high-fat diet was administered only in the case of the administration of the Jeju liposome extract in addition to the high-fat diet for the observation of the tissue sections of the extracted experimental animals. The group shows significant progression of fatty liver.

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

In the case of p -coumaric acid isolated from Jeju jeju extract, the concentration of SREBP-1c (sterol regulatory element binding protein-1c) was inhibited in HepG2 cells induced fat accumulation. 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 experimental results can be said that the Jeju jeoridae extract can be useful for fatty liver improvement.

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.

Also in the fatty liver improver composition of the present invention, in relation to the meaning of the jeju jeoldae extract, its effective amount and the like, the above-described bar as described above with respect to the obesity improver composition of the present invention is effective.

On the other hand, fatty liver may progress to hepatitis, cirrhosis, liver cancer and the like if left untreated. Therefore, the fatty liver improver composition of the present invention may be used for the purpose of preventing hepatitis, cirrhosis or liver cancer caused by fatty liver.

Therefore, in another aspect, the present invention can be understood as a composition for the prevention of hepatitis, liver cirrhosis or liver cancer, which is a disease caused by fatty liver containing jejudae extract as an active ingredient.

In another aspect, the present invention relates to an insulin resistance improving agent composition comprising jeju jeoldae extract or p -coumaric acid as an active ingredient.

Obesity is one of several causes of insulin resistance. Although insulin resistance does not develop at high obesity, insulin resistance and obesity have a close correlation. In general, the more severe the obesity, the more visceral obesity is known to increase the insulin resistance.

Adipose cells secrete the bioactive adipocytokine, which plays an important role in maintaining metabolic homeostasis, but when obesity, ie, excessive accumulation of fat, adipocytokine production and secretion It is understood that insulin resistance is caused by excess or underestimation resulting in a breakdown of the balance.

Among the adipocytokines, there are those that promote insulin sensitivity and cause insulin resistance. The former are adiponectin and AMPK (AMP-dependent protein kinase), and the latter are representative of TNF-α, lL-6, and resistin. (resistin) etc. are mentioned.

In particular, studies have shown that a decrease in AMPK activity involves insulin resistance and hyperglycemia, and thus, a substance capable of activating AMPK may be a candidate substance for improving insulin resistance (J. Biol. Chem., 277 (28). ): 25226-32, 2002; Diabetes. 51 (7): 2074-81, 2002; J. Clin. Invest. 108 (8): 1167-74, 2001).

The following Examples and Experimental Examples show that the jeju jeoldae extract or p -coumaric acid isolated therefrom, which is an active ingredient of the obesity improver composition of the present invention, activates AMPK. These experimental results can be said that the jeju jeoldae extract can be useful as not only an obesity improver but also an insulin resistance improver.

In the present specification, the term "insulin resistance" refers to a state in which cells, organs, human bodies, etc. require more than a normal amount of insulin for normal metabolic activity, that is, a state of insufficiency of insulin, in which insulin has a decreased ability to act. It is taken in the same sense as non-dependent diabetes mellitus or type 2 diabetes. Diabetes is an insulin-dependent diabetes mellitus (type 1 diabetes) that requires insulin for its treatment as a result of the destruction of β-cells in the pancreas, and insulin-independent diabetes mellitus, which does not require insulin for its treatment. Type 2 diabetes), insulin resistance, insulin independent diabetes and type 2 diabetes are taken in the same sense in the art.

Also in the insulin resistance improving agent composition of the present invention, as described in connection with the meaning of the jeju jeoldae extract, the effective amount thereof, and the like as described above with respect to the obesity improving composition of the present invention as it is effective.

Obesity improver composition, diet composition, hyperlipidemia improver composition, composition for preventing obesity complications, fatty liver improver composition, preventive composition for diseases caused by fatty liver and insulin resistance improver composition (hereinafter "composition of the present invention") In a specific aspect, it can be grasped | ascertained as a food composition.

The food composition of the present invention may be prepared as a dietary supplement, a special nutritional supplement, 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.

As the physiologically active substance, catechins such as catechin, epicatechin, gallocatechin, epigallocatechin, vitamins such as retinol, ascorbic acid, tocopherol, calciferol, thiamine, riboflavin, and the like can be used.

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 is a powder derived from any natural product already known to have an obesity improving activity, hangover relieving activity, etc. in order to enhance flavor or palatability, enhance its functionality, or add other functionalities (such as hangover relieving activity). Or extracts, such as ointment powder or extract, bean sprout powder or extract, clam powder or extract, oyster powder or extract, hawthorn powder or extract, radish juice or extract, cucumber juice or extract, leek juice or extract, spinach Juice or extract, lotus root juice or extract, sesame juice or extract, pine needle juice or extract, ginseng juice or extract, birch sulphate powder or extract, licorice powder or extract, brownish powder or extract, reed powder or extract, sine powder or extract, Gourd powder or extract, ginger powder or extract, jujube powder or Silver extract, phosphorus powder or extract, earth powder or extract, hydrophobic powder or extract, white extract powder or extract, spirit powder or extract, dermis (peel of tangerine) powder or extract, wolfberry powder or extract, green tea powder or extract, Schizandra powder or extracts, hawthorn powder or extracts, borage powders or extracts, old root powder or extracts, cinnamon powder or extracts, decosinol and the like can be exemplified. 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.

The composition of the present invention may be regarded as a pharmaceutical composition in another specific embodiment.

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).

As used herein, "pharmaceutically acceptable" means that the subject of application (prescription) does not have more toxicity (adequately less toxic) than is applicable without inhibiting the activity of the active ingredient.

Examples of pharmaceutically acceptable carriers include lactose, glucose, sucrose, starch (such as corn starch, potato starch, etc.), cellulose, derivatives thereof (such as sodium carboxymethyl cellulose, ethylcellulose, etc.), malt, gelatin, talc, Solid lubricants (such as stearic acid, magnesium stearate, etc.), calcium sulfate, vegetable oils (such as peanut oil, cottonseed oil, sesame oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, etc.), alginic acid, emulsifiers (such as TWEENS), wetting agents (Such as sodium lauryl sulfate), colorants, flavors, tableting agents, stabilizers, antioxidants, preservatives, water, saline, phosphate buffer solutions, and the like. 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. Suitable excipients when prepared in an injection solution may include Ringer's solution, isotonic sodium chloride, and the like.

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, the present invention can provide an obesity improving composition. Also, according to the present invention, a composition for improving liver fat remedy can be provided. In addition, according to the present invention can provide a hyperlipidemic improver composition, insulin resistance improver composition and the like. Obesity enhancer composition of the present invention may be commercialized as a functional food, drugs and the like.

Figure 1 shows the chromatogram of the jeju jeoldae hot water extract and the molecular structure of the main component.
2 is the weight of the experimental animals (HFD + SQE) when fed a normal diet (ND), fed a high-fat diet (HFD) and oral administration of high-fat diet and jejudae extract. weight) is measured every 5 days for 70 days.
Figure 3 of the experimental animals fed a normal diet (ND), fed a high-fat diet (HFD) and orally administered high-fat diet and jejudae extract (HFD + SQE) of the experimental animals after 70 days This is a result of checking the cell size of epididymal adipose tissue.
FIG. 4 shows that the normal animals were fed the normal diet (ND), the high fat diet was fed (HFD), and the high fat diet and Jeju chorus extract were orally administered (HFD + SQE) of the experimental animals after 70 days. This is a result of measuring RNA expression of adiponectin in abdominal fat.
FIG. 5 shows that when the normal diet was fed to the experimental animals (ND), the high fat diet was fed (HFD), and the oral administration of the high fat diet and Jeju chorus extract (HFD + SQE) 70 days after This is the result of measuring protein expression levels of p-AMPK and p-ACC in abdominal fat.
FIG. 6 shows that when the normal diet was fed to the experimental animals (ND), the high-fat diet was fed (HFD) and the oral administration of the high-fat diet and Jeju sap extract (HFD + SQE) 70 days after Liver sections were observed under a microscope.
Figure 7 is the result of measuring the protein expression of p-AMPK, p-ACC in differentiated 3T3-L1 adipocytes of Jeju jejudae extract (SQE).
Figure 8 is the result of measuring the protein expression of p-AMPK, p-ACC in differentiated L6 skeletal muscle cells of Jeju jejudae extract (SQE).
Figure 9 is the result of measuring the protein expression of p-AMPK, p-ACC in HepG2 liver cells induced localization of Jeju jejudae extract (SQE).
Figure 10 is the result of measuring the RNA expression of CPT-1a in differentiated 3T3-L1 adipocytes of Jeju jejudae extract (SQE).
11 shows that p -coumaric acid inhibits the accumulation of triglycerides in differentiated 3T3-L1 adipocytes.
FIG. 12 shows that p -coumaric acid increases LKB1 phosphorylation, AMPK phosphorylation and ACC phosphorylation in a concentration-dependent manner in differentiated 3T3-L1 adipocytes.
FIG. 13 shows that p -coumaric acid increases phosphorylation of AMPK and ACC concentration-dependently in differentiated L6 skeletal muscle cells.
14 shows that p -coumaric acid increases PPARα expression in a concentration dependent manner in differentiated L6 skeletal muscle cells.
FIG. 15 shows that p -coumaric acid increases phosphorylation of AMPK and ACC concentration-dependently in HepG2 cells induced with fat accumulation.
FIG. 16 shows that p -coumaric acid increases SREBP-1c expression in a concentration-dependent manner in HepG2 cells induced with 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 Joridae Extract and Isolation of Bioactive Materials

<Example 1> Preparation of Jeju Sasa extract Example 1

6 kg of Jeju cooking rod was washed with tap water, lyophilized and ground. The pulverized Jeju chopsticks were immersed in water to extract hot water at 90 ° C. for 4 hours, and then concentrated under reduced pressure to remove the extraction solvent to obtain the extract of Jeju chopsticks in powder form.

<Example 2> Preparation Example 2 of Jeju Joridae extract

6 kg of Jeju cooking rod was washed with tap water, lyophilized and ground. Grinded jeju bar was added to water containing 3% of its weight in viscozyme ™ (Novo Nordisk, Denmark), 3% celluclast ™ (Novo Nordisk, Denmark) and citric acid (pH 4.5) and then for 50 to 5 hours. Extract and deactivate the enzyme at 100 ° C. for 1 hour. After concentration under reduced pressure, lyophilization was used to remove the extraction solvent, and the extract of Jeju sapling powder was prepared.

Example 3 Isolation of Bioactive Substance

(1) Fraction of Jeju Joridae Extract

100 g of Jeju jejudae extract was suspended in 1 L of distilled water, and then, n-hexane, EtOAc, and 1 L of n-BuOH were added thereto and extracted three times in succession, and then concentrated at 40 ° C. using a rotary concentrator.

(2) Preparation of Standard Solution

As a standard solution for the determination of the components of the jejudae hot water extract, 10 mg of chlorogenic acid, p -coumaric acid, and tricin were dissolved in 1 mL of DMSO (dimethylsulfoxide), diluted with 9 mL of methanol, and used as a standard solution.

(3) Preparation of test solution

Take about 500 mg of the jeju jeori hot water extract of <Example 1>, add 2.5 mL of DMSO, add 2.5 mL of methanol, sonicate for 10 minutes, and then extract the extract with 0.50 mm Teflon (PTFE, polytetrafluoroethylene) and a syringe filter (Advantec Toyo Roshi Kaisha). Ltd., DISMIC ^R -13 _JP , Japan) and used as a test solution.

(4) Analysis of the compound

Qualitative analysis of Jeju hot water extract was performed using HPLC (Waters, 2695 Alliance system, USA), Sunfire ^™ C ₁₈ column (250 × 4.6 mm ID. 5 μm), and the column temperature was maintained at 40 ° C. Component detection of the jeju jeoldae fraction was detected in UV absorbance in the range of 200 ~ 600 nm using a PDA detector, and the mobile phase was separated by gradient elution using MeOH and ultrapure water (Table 1). The flow rate was maintained at 1.0 mL / min.

(5) Separation and Identification Method of Compound

The equipment for separating the Jeju hot water extract compound was used HPLC (Waters, 2695 Alliance system, USA), Symmetryprep ^TM C ₁₈ column (7.8 300 mm ID. 7 m), the column temperature was maintained at 40 ℃. The detector was detected at UV absorbance in the range of 200-600 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 the same peak was repeatedly collected. Was performed using the NMR (Nuclear Magnetic Resonance) spectroscopy is a 400 MHz FT-NMR (JEOL Co., JNM-LA, Japan) used for structure analysis, the measurement solvent was used as Merck's CD ₃ OD and CDCl _3.

(6) Identification of Isolated Compounds

Three compounds contained in the jejudae hot water extract were isolated using HPLC and 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 ., 2010, 31 (4), 1088-1090) acid, p -coumaric acid and tricin were identified as compounds. In addition, qualitative analysis was performed using a standard of each separated compound to confirm this. The Triple injected at 320nm revealed the retention of each compound peak, as a result chlorogenic acid peak is p close to 7 minutes in order to check the configuration compound isolated from the hot-water extract - between Kumar acid peak is 10-11 minutes tricin Peaks were detected between 18 and 19 minutes and showed the same retention and absorbance as the standard solution. Chlorogenic acid, p -coumaric acid, and tricin were found to contain 0.132, 23.706, and 0.286 mg, respectively, in Jeju extract. As a result of analyzing the major components of the hot water extract of Jeju jejudae, p -coumaric acid showed the highest content. Therefore, the following experiments investigated the effect of p -coumaric acid, a major component of hydrothermal extract, on cell lipid metabolism.

< Experimental Example > Jeju jejudae extract and isolated from it p -Metabolic Disease Improvement Activity Test of Kumaric Acid

< Experimental Example  1> Metabolic Disease Improvement Activity Experiment

Experimental Example 1-1 Body Weight and Body Fat Measurement Experiment in Animal Experiment

Four-week-old C57BL / 6 male mice purchased from Nara Biotech Co., Ltd. were adapted for 1 week with solid feed and water, and randomly divided mice of similar weight into three groups of 10 animals each. The normal group (ND) is a normal diet, the obese control group (HFD) is a high-fat diet, the experimental group (HFD + SQE) is a high-fat diet and the Jeju jeju extract (150mg / kg of body weight / day) orally administered two dogs Separated and bred. The breeding environment was maintained at a temperature of 23 ° C., a humidity of 50 ± 5%, and a night and day cycle for 12 hours.

In the normal diet, a 10% kcal fat diet (Research Diets, USA) feed was used, and a high fat diet was used a 60% kcal fat diet (Research Diets, USA) feed.

The effect of jejudae extract on body weight was measured every 70 days using an animal weight scale at the same time period, and the experimental results are shown in the graph in FIG. 2, and the dietary intake was measured every 5 days for 70 days at the same time period for 5 days. The average (g / cage / 5day) of the unit measurand is shown in Table 2. And the total weight gain for 70 days (measured by subtracting the initial weight from 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 * between a, b and c has statistical significance ( p <0.05)

Weight gain total division ND HFD HFD + SQE Weight gain (g) 5.65 ± 0.60 ^a 15.60 ± 0.78 ^c 10.76 ± 0.59 ^b * Between a, b and c have statistical significance ( p <0.05)

2 and Table 3 show that the weight gain was significantly decreased in the HFD + SQE group administered with Jeju jejudae extract compared to the HFD group. In Table 2 it can be seen that the HFD + SQE group did not have a small dietary intake compared to the HFD group.

In order to confirm the effect of Jeju jejudae extract on the weight of abdominal fat (white fat) and kidney fat, after anesthesia with 70 days of anesthesia, the weight was extracted by anesthesia, and the results are shown in Table 4. , HFD + SQE showed less white fat weight than HFD group.

Increased total amount of abdominal 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 * between a, b and c has statistical significance ( p <0.05)

On the other hand, in order to confirm the effect of the jeju jeoldae extract on the cell size of the abdominal fat (epididymal adipose tissue), after an experiment for 70 days in Experimental Example 1 was anesthetized with ether and extracted to determine the size of the cells after tissue sections Observation was made under a microscope, and the results of the experiment are shown as a photograph of tissue sections of two individuals per group. 3 shows that the adipose tissue cell size of the HFD + SQE group was similar to that of the ND group, whereas the HFD group significantly increased the adipose tissue size.

Experimental Example 1-2 Adiponectin Expression Measurement by RT - PCR

In order to confirm the effect of Jeju jeju extract on the expression of cytokines in the abdominal fat (epididymal adipose tissue), after an experiment for 70 days in Experiment 1 after anesthesia with an ether extracted and real RNA separation after real RNA separation It was measured by the PCR technique, and the experimental results are shown graphically in FIG.

For total RNA for real-time PCR, add 500 µl of Trizol Reaent (Invitrogen, USA) to homogenize cells for 5-10 minutes at room temperature, transfer to a tube, and centrifuge by adding 100 µl of chloroform (12,000 × g, 15). Minutes). The supernatant was recovered, transferred to a new tube, and the same amount of isopropanol was added and centrifuged (12,000 x g, 15 minutes) to precipitate RNA. The precipitated RNA was washed three times with 75% ethanol, dried, dissolved in DEPC treated distilled water, and quantified by measuring absorbance at 260 nm using a nanodrop 2000 spectrometer (Nanodrop, USA). An RNA sample having a value in the ratio of A260 / A280 nm in the range of 1.8-2.2 was treated with DNase (Wako, Japan) to separate only pure RNA and used for the experiment.

cDNA was synthesized using Maxime RT PreMix Kit (iNtRON Biotechnology, Korea). 1µg total RNA and nuclease-free water were added to the tube to a total of 20µl, and the reaction was carried out at 45 ° C. for 60 minutes and at 95 ° C. for 5 minutes to synthesize cDNA, which was diluted by adding 30 µl of nuclease-free water. Then used for PCR.

Real time polymerase chain reaction (PCR) was performed at 5 µl cDNA, each primer at 0.4 µl (10 pmol / L / µl), 2 µl nuclease-free water and 6 µl iQ ™ SYBR? Green Supermix (Bio-rad, USA) was mixed and then performed using a Peltier thermal cycler (Bio-rad, USA) real time PCR instrument. The PCR conditions were amplified 49 times with 95 ℃ / 20sec, 65 ℃ / 20sec, 72 ℃ / 30sec, and the results were measured for each amplification. Was performed to analyze the melting curve of the primer. Results were analyzed using Chromo4 Real-Time PCR Detection System v1.10 (Bio-rad, USA) to quantify relative values of β-actin. The base sequences of the primers used in 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: 2). ); β-actin, 5'-AGG CTG TGC TGT CCC TGT AT-3 '(SEQ ID NO: 3) and 5'-ACC CAA GAA GGA AGG CTG GA-3' (SEQ ID NO: 4).

4 shows that the expression level of adiponectin in the HFD group was significantly reduced compared to the ND group, whereas the expression level of adiponectin in the HFD + SQE group was significantly increased compared to the obesity control group.

Experimental Example 1-3 Western Experiment for measuring the expression level of p - AMPK and p- ACC through blot

In order to confirm the effect of Jeju jerk extract on the AMPK activation of abdominal fat (white fat), after an experiment for 70 days in Experimental Example 1 was anesthetized with ether and extracted and measured by Western blot method after protein separation. The experimental results are shown in the photograph 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 concentration was measured and absorbed at 595 nm with a micro reader using a Bio-rad protein assay kit (Bio-rad, USA). 35 ㎍ of protein was separated by electrophoresis on 10% SDS-polyacrylamide gel and then transferred (100V, 120 min) to poly vinylidene difluoride (PVDF) membrane (Milipore, USA). Protein-transferred PVDF membrane was blocked with Tris buffered saline (TTBS) solution containing 0.1% Tween 20 containing 5% skim milk or 5% BSA at room temperature for 1 hour, followed by primary antibody (p-AMPK, p-ACC, and β-actin). p-AMPK antibody (1: 2,000, Cell Signaling, USA), p-ACC antibody (1: 1,000, Cell Signaling, USA), and β-actin antibody clone AC-74 (1: 10,000, Sigma, USA) Was carried out at 4 ° C. overnight. After the first antibody reaction, the membrane was washed with 0.1% TTBS solution and then reacted for 1 hour by diluting the peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, USA) to 1: 2,000, 1: 5,000 or 1: 10,000. Washed with TBS-T. Expression of each protein was detected by X-ray film (Ortho CP-G plus; Agfa Gevaert NV, Belgium) by reacting with a WEST-ZOL western blot detection system (iNtRON Biotechology, Korea).

5 shows that the AMPK activation (p-AMPK) and ACC inactivation (p-ACC) of the HFD group is significantly reduced compared to the ND group, while AMPK activation and ACC inactivation of the HFD + SQE group are HFD It can be seen that significantly increased compared to the group.

Experimental Example 1-4 Blood Total Cholesterol and Blood Triglyceride Test

The effect of Jeju choridae extract on the total cholesterol and blood triglyceride content in blood was measured using a kit for analysis (Asan Pharmaceutical, Korea) after separating the serum after 70 days in Experimental Example 1 It is shown in Table 5, the HFD + SQE group has a lower total blood cholesterol and triglyceride content than the HFD group.

Total increase in total cholesterol (T-CHO) and triglyceride (TG) in blood 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 * between a, b and c has statistical significance ( p <0.05)

Experimental Example 1-5 Liver Tissue Observation Experiment

The effect of Jeju jejudae extract on liver tissue damage was confirmed by anesthesia after extraction with ether after an experiment for 70 days in Experimental Example 1 through a microscopic observation after tissue section, the experimental results of two individuals per group in FIG. Tissue sections are shown. FIG. 6 shows that the HFD + SQE group is similar to the ND group, and the HFD group shows an increase in the amount of fat globules compared with the HFD + SQE group or the ND group.

<Experimental Example 1-6> Experiment for Measuring Liver Damage Indicator Enzyme Activity

GPT, GOT, and LDH levels, which are used as indicators of liver tissue damage, were measured using an analysis kit (Asan Pharmaceutical, Korea) after separation of serum after 70 days in Experimental Example 1. The results are shown in Table 6. Referring to Table 6, the HFD + SQE group showed lower GPT, GOT, and LDH levels as well as the ND group values, compared to the HFD group.

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 * between a, b and c has statistical significance ( p <0.05)

Experimental Example 1-7 Western in Cell Experiment Experiment for measuring expression level of p- AMPK through blot

In order to confirm the results of activating AMPK at the cellular level, the extracts of Jeju jejudae were cultured by culturing 3T3-L1 cells (fat cells), L6 cells (skeletal muscle cells), and HepG2 cells (hepatocytes). 7, 8, and 9 are respectively shown in the photograph.

To analyze the effects of AMP-activated protein kinase (AMPK) and its substrate, Acetyl Co-A carboxylase (ACC), on phosphorylation and expression of its subgenes, in differentiated adipocytes, It was performed as follows. 3T3-L1 progenitor cells were inoculated into 6 well cell culture plates (1.6 or 2.0 × 10 ⁵ cells / well) using DMEM medium supplemented with 10% BCS and 1% P / S. to be confluent. In the pre-confluent state, the medium was changed once more and cultured for 48 hours. In the confluent state (day 0, differentiation induction), the culture medium was differentiated to induction 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 -1-methylxanthine (IBMX; Sigma, USA) and 5 μg / ml insulin (Sigma, USA)] was induced to differentiate for 2 days (day 2 of induction of differentiation). After 2 days (day 4 of differentiation), the cells were exchanged with DMEM medium containing 10% FBS and 1% P / S every 2 days until used in the experiment. After 8-10 days of differentiation, the medium was removed from the culture vessel and washed twice with DMEM, followed by 16 hours of incubation with serum-free medium (0.5% BSA + DMEM), followed by concentration (20, 100, 250, 500 and 1,000 ㎍). / Ml) for 24 hours to confirm the phosphorylation of AMPK and ACC.

In order to analyze the effects of AMP-activated protein kinase (AMPK) and phosphorylation of Acetyl Co-A carboxylase (ACC), the substrate of Jeju jeju extract on L6 myotube, the differentiation of muscle cells was performed as follows. L6 myoblasts were inoculated into 12 well cell culture plates (1 × 10 ⁵ cells / well) using DMEM medium containing 10% FBS and 1% P / S and incubated for 48 hours to achieve pre-confluent status. In the pre-confluent state, medium (DMEM with 10% FBS) was exchanged once more and cultured for 48 hours. In the confluent state (day 0 differentiation), differentiation was induced by exchanging the culture medium with differentiation induction medium (DMEM medium with 2% FBS). The medium was changed to the same medium every two days until used for the experiment. On 9-10 days of differentiation, the medium was removed from the culture vessel, washed with DMEM medium, and incubated for 4 hours with DMEM medium added with 0.5% BSA. Then, the concentration of (125, 250, 500 and 1,000 ㎍ / ㎖) was treated for 24 hours to confirm the phosphorylation of AMPK and ACC.

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

Samples treated cells were washed twice with PBS for Western blot analysis, followed by 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. The protein was separated by vortexing for 1 hour using a protein separation reagent containing a buffer (Upstate Biotechnology, USA), followed by centrifugation (15000 rpm, 4 ° C., 20 minutes) to obtain a supernatant. Protein concentration was measured and absorbed at 595 nm with a micro reader using a Bio-rad protein assay kit (Bio-rad, USA). 35 ㎍ of protein was separated by electrophoresis on 10% SDS-polyacrylamide gel and then transferred (100V, 120 min) to poly vinylidene difluoride (PVDF) membrane (Milipore, USA). Protein-transferred PVDF membrane was blocked with Tris buffered saline (TTBS) solution containing 0.1% Tween 20 containing 5% skim milk or 5% BSA at room temperature for 1 hour, followed by primary antibody (p-AMPK, AMPK, p-ACC, ACC and β-actin). p-AMPK antibody (1: 2,000, Cell Signaling, USA), AMPKα antibody (1: 5,000, Cell Signaling, USA), p-ACC antibody (1: 1,000, Cell Signaling, USA), ACC antibody (1: 1,000, Cell Signaling, USA) and β-actin antibody clone AC-74 (1: 10,000, Sigma, USA) was performed overnight at 4 ℃. After the first antibody reaction, the membrane was washed with 0.1% TTBS solution and then reacted for 1 hour by diluting the peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, USA) to 1: 2,000, 1: 5,000 or 1: 10,000. Washed with TBS-T. Expression of each protein was detected by X-ray film (Ortho CP-G plus; Agfa Gevaert NV, Belgium) by reacting with a WEST-ZOL western blot detection system (iNtRON Biotechology, Korea).

7, 8 and 9 it can be seen that the jejudae extract increases AMPK activity in all three cell types.

Experimental Example 1-8 CPT- 1a Expression Measurement by RT - PCR

In particular, it was confirmed that the RNA extract of the AMPK sub-regulator CPT-1a, which is an indicator measuring the β-oxidation degree of fatty acids in 3T3-L1 cells, and the experimental results are shown graphically in FIG. 9. Referring to Figure 10 it can be seen that jeju jeoldae extract increases the expression of CPT-1a.

For total RNA for real-time PCR, add 500 µl of Trizol Reaent (Invitrogen, USA) to homogenize cells for 5-10 minutes at room temperature, transfer to a tube, and centrifuge by adding 100 µl of chloroform (12,000 × g, 15). Minutes). The supernatant was recovered, transferred to a new tube, and the same amount of isopropanol was added and centrifuged (12,000 x g, 15 minutes) to precipitate RNA. The precipitated RNA was washed three times with 75% ethanol, dried, dissolved in DEPC treated distilled water, and quantified by measuring absorbance at 260 nm using a nanodrop 2000 spectrometer (Nanodrop, USA). An RNA sample having a value in the ratio of A260 / A280 nm in the range of 1.8-2.2 was treated with DNase (Wako, Japan) to separate only pure RNA and used for the experiment.

cDNA was synthesized using Maxime RT PreMix Kit (iNtRON Biotechnology, Korea). 1μg total RNA and nuclease-free water were added to the tube to a total of 20μl, reacted for 5 minutes at 45 ℃ 60min, 95 ℃, synthesized cDNA, and diluted with 30μl of nuclease-free water added thereto. Used for PCR.

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

< Experimental Example  2> p -Metabolic Disease Improvement Activity Test of Kumaric Acid

Experimental Example 2-1 Experiment of Inhibition of Differentiation of Progenitor Cells into Adipocytes

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

After two more days of incubation, the cells were filled with DMEM medium containing 1% PS, 10% fetal bovine serum (FBS), MDI (0.5 mM isobutylmethylxanthine (IBMX), 1M dexamethasone, and 1 μg / mL insulin). Differentiation was induced by addition. Two days after the induction of differentiation, the cells were replaced with DMEM medium containing 1% PS, 10% FBS, and 1 μg / mL insulin. After two days, the cells were replaced with DMEM medium containing 1% PS and 10% FBS. The medium was changed every two days until the experiment.

During the induction of adipocyte differentiation, samples ( p -coumaric acid) were treated with each medium at a concentration of 100 uM, and the degree of adipocyte differentiation was observed on day 8, when the differentiation was completed. Specifically, 3T3-L1 progenitor cells were washed twice with PBS (Phosphate-buffered saline) 8 days after induction of differentiation and fixed with 3.7% formaldehyde for 1 hour. After washing twice with distilled water and then treated with 0.6% Oil Red O reagent was stained for 1 hour. After staining, oil red O was removed, washed twice with distilled water first, and the cells were dried. 4% Nonidet P-40 was added to quantify the stained fat droplets. Since the numerical value was measured at the absorbance of 540nm, the results are shown by comparing the absorbance of the pure differentiated cells and the absorbance of the cells treated with the sample.

The results are shown in FIG. Referring to FIG. 11, it can be seen that the TG content was significantly decreased in the p -coumaric acid treated group during Day4 to Day8. This effect may be inferred that p -coumaric acid is associated with lipolysis and TG synthesis after adipocyte differentiation.

Experimental Example 2-2 Western in Differentiated 3 T3 - L1 Adipocytes LKB1 p- and p-AMPK expression level measured in the experiment with the blot

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

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

The results are shown in FIG. Referring to Figure 12, p- coumaric acid increased the phosphorylation of LKB1 known to activate AMPK in differentiated 3T3-L1 cells, it was confirmed that the phosphorylation of AMPK and ACC also increased. These results suggest that p -coumaric acid inhibits lipid accumulation in adipocytes and has anti-obesity effects by activating lipid metabolism such as β-oxidation via AMPK signal in mature adipocytes.

Experimental Example 2-3 Western in Differentiated L6 Cells Experiment for measuring the expression level of p - AMPK and p- ACC through blot

Rat derived L6 skeletal muscle cell line was purchased from ATCC. Cells were incubated in DMEM containing 10% FBS and 1% PS and maintained at 37 ° C. in 5% CO ₂ conditions. Cell differentiation was replaced with DMEM medium containing 2% FBS every other day before use in experiments. Differentiated L6 cells were fasted for 4 hours with serum-free DMEM containing 0.5% bovine serum albumin (BSA), and then treated with p-coumaric acid for 48 hours.

Next, the phosphorylation degree 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. Referring to FIG. 13, it can be seen that the phosphorylation of AMPK and ACC of the group treated with p -coumaric acid was increased in a concentration-dependent manner compared to the control group. In particular, AMPK and ACC showed a significant increase in phosphorylation at the highest concentration of 50 uM. 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 reported to ultimately 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 Western in Differentiated L6 Cells PPAR α expression level measurement experiment through blot

In the same manner as in <Experimental Example 2-3>, p -coumaric acid was treated by concentration for 48 hours, and the expression level of PPARα was analyzed by Western blot in the same manner as in <Experimental Example 2-2>.

The results are shown in FIG. Referring to FIG. 14, it can be seen that PPARα expression was increased in the group treated with p -coumaric acid as compared to PPARα expression in the control group. Peroxisome proliferator-activated receptors (PPARs) are known to regulate gene expression related to lipid metabolism and energy homeostasis (Diabetes, 52: 2249-2259, 2003). In particular, PPAR plays an important role in the transcriptional regulation of genes related to β-oxidation, a process of producing energy using fatty acids (Molecular and cellular biology, 20 (5): 1868-1876, 2000). The increased expression of PPARα, 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 Western in HepG2 Cells Experiment for measuring the expression level of p - AMPK and p- ACC through blot

HepG2 cell line was purchased from ATCC. Cell culture was incubated in 37 ℃, 5% CO ₂ conditions using DMEM medium added 1% PS to 10% FBS. Cells were maintained by subculture when the cells were 80% full at the bottom of the T-75 cell culture flask. Induction of fat 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 6 well cell culture plates (1 × 10 ⁶ cells / well) using DMEM medium supplemented with 10% FBS and 1% PS and 80% cold on the bottom of the 6 well cell culture plate. Incubated until. Starvation overnight was performed in DMEM medium containing 0.5% BSA with 80% cold cells. The free fatty fatty acid oleic acid was exchanged with 600uM and p -coumaric acid for 24 hours and incubated for 24 hours to induce fat 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 Figure 15 it can be seen that the concentration-dependent increase in AMPK, ACC phosphorylation involved in fatty acid oxidation.

Experimental Example 2-5 Western in HepG2 Cells expression of SREBP -1c through the measurement blot experiments

In the same manner as in <Experimental Example 2-4>, oleic acid 600M and p -coumaric acid were incubated for 24 hours in cells in which fat accumulation was induced, followed by SREBP- in the same manner as in <Experimental Example 2-2>. Expression level of 1c was analyzed by Western blot.

The results are shown in FIG. Referring to Figure 16 it can be seen that the concentration-dependent decrease in SREBP-1c involved in the synthesis of fatty acids and triglycerides.

Claims (20)

Obesity improving composition comprising jeju jeoldae extract or p -coumaric acid as an active ingredient.
The method of claim 1,
The extract is an obesity improver composition, characterized in that obtained by using water, ethanol or a mixed solvent thereof.
The method of claim 1,
The composition is an obesity improver composition, characterized in that the food composition.
The method of claim 1,
The composition is an obesity improver composition, characterized in that the pharmaceutical composition.
Hyperlipidemia improver composition comprising jeju jeoldae extract or p -coumaric acid as an active ingredient.
The method of claim 5,
The extract is hyperlipidemic composition, characterized in that obtained using water, ethanol or a mixed solvent thereof.
The method of claim 5,
The composition is a hyperlipidemic improver composition, characterized in that the food composition.
The method of claim 5,
The composition is a hyperlipidemic improver composition, characterized in that the pharmaceutical composition.
Fatty liver improving composition comprising jeju jeoldae extract or p -coumaric acid as an active ingredient.
10. The method of claim 9,
The extract is a fatty liver improver composition, characterized in that obtained by using water, ethanol or a mixed solvent thereof.
10. The method of claim 9,
The composition is a fatty liver improver composition, characterized in that the food composition.
10. The method of claim 9,
The composition is a fatty liver improver composition, characterized in that the pharmaceutical composition.
Insulin resistance improving composition comprising jeju jeoldae extract or p -coumaric acid as an active ingredient.
The method of claim 13,
The extract is insulin resistance improving composition, characterized in that obtained by using water, ethanol or a mixed solvent thereof.
The method of claim 13,
The composition is an insulin resistance improver composition, characterized in that the food composition.
The method of claim 13,
The composition is an insulin resistance improver composition, characterized in that the pharmaceutical composition.
Food composition for diet comprising jeju jeoldae extract or p -coumaric acid as an active ingredient.
18. The method of claim 17,
The extract is a food composition for a diet, characterized in that obtained by using water, ethanol or a mixed solvent thereof.
As a food composition for the prevention of diseases caused by obesity containing jeju jeoldae extract or p -coumaric acid as an active ingredient,
The obesity complications are hypertension, elevated blood cholesterol, kidney disease, stroke, arteriosclerosis, fatty liver, arthritis, cancer, sleep apnea and diabetes, food composition for preventing diseases caused by obesity, characterized in that one of the causes.
Food composition for the prevention of hepatitis, liver cirrhosis or liver cancer, which is a disease caused by fatty liver containing jeju jeoldae extract or p -coumaric acid as an active ingredient.

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