KR20120096222A - Composition for improving obesity and fatty liver - Google Patents

Abstract

PURPOSE: A composition containing Gracilaria vermiculophylla (Ohmi) Papenfuss extract or fraction thereof is provided to suppress differentiation of 3T3-L1 preadipocytes to adipocytes. CONSTITUTION: A composition for treating obesity contains Gracilaria vermiculophylla or, (Ohmi) Papenfuss extract, hexane fraction or ethyl acetate fraction thereof as an active ingredient. The extract is prepared using water, ethanol, or mixture solvent thereof. The composition is a food composition or pharmaceutical composition. A composition for treating hyperlipidemia and fatty liver contains the extract or fraction as an active ingredient.

Description

Composition for Improving Obesity and Fatty Liver

The present invention relates to obesity and fatty liver improver composition.

Recently, the obese population is rapidly increasing due to the improvement of living standards according 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) believes that the Asian Standard Body Mass Index (BMI) is 23–25 overweight, 25–30 obese, and over 30 highly obese.

The causes of obesity include high fat and high calorie diet, lack of exercise due to busy social environment, and endocrine disorders. Among them, 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 is Gracilaria vermiculophylla (Ohmi) Papenfuss extracts and fractions initiate the activity of obesity and fatty liver.

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.

The inventors of the present invention, as confirmed in the following examples and experimental examples, sesame seed 80% ethanol extract, its hexane fraction or ethyl acetate fraction to induce differentiation in 3T3-L1 precursor adipocytes (IBMX, DEXA., Insulin) When treated with, concentration-dependently reduced formation of fat droplets, and concentration-dependently decreased expression of peroxisome proliferator-activated receptor γ (PPAR), CCAAT / enhancer binding protein α (C / EBPα), and aP2. Sikkim could be confirmed. In particular, the activity of the ethyl acetate fraction was the best.

PPARγ and C / EBPa are known as transcription factors that induce differentiation of adipocytes, and aP2 is known as an adipocyte differentiation indicator protein (Genes De 2000, 14 (11) 1293 ~ 1307; Physiol Rev 1998, 78 ( 3): 783 ~ 809; Annu Rev Biochem 2008, 77: 289 ~ 312; Genes Dev 1996, 10: 1096-1107).

As a result of examining the effect on the AMPK (AMP-activated protein kinase) signal transduction of the ethyl acetate fraction having the highest activity among the extracts and fractions, the fraction activates LBK1 (AMPK-Kinase) and the ROS (reactive oxygen species) AMPK was activated to inhibit the activity of acetyl-CoA carboxylase (ACC) and uncoupling protein 2 (UCP2) and carnitine palmitoyl transferase-1α (CPT-1α).

AMPK is an enzyme that plays a role as a sensor for maintaining energy homeostasis in cells and is known to be 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). The activation of AMPK is known to be involved in LKB1 (AMPK-Kinase), a regulated phosphatase (Curr Biol. 13 (22): 2004-2008, 2003). When AMPK is activated, ACC is phosphorylated and inactivated. Is an enzyme involved in the early stages 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 AMPK is activated, it is possible to suppress the accumulation of fat in the body, and as a result, to suppress the increase in body weight, and also to reduce the concentration of triglycerides and cholesterol in the blood.

The obesity improver composition of the present invention is provided based on the experimental results as described above, and the obesity improver composition of the present invention is characterized in that it contains an extract of kohlrabi, its hexane fraction, or an ethyl acetate fraction as an active ingredient. .

In the present specification, the "Corntail Extract" is used as an extractant, and the lower alcohol having 1 to 4 carbon atoms such as water, methanol, ethanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl It means an extract obtained using acetate, butyl acetate, dichloromethane, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol or a mixed solvent thereof. 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. 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. The "tailed kohlrabi extract" means preferably obtained by using water, ethanol or a mixed solvent thereof as an extraction solvent.

In addition, in the present specification, the "hexane fraction of the sesame seed extract" refers to the sesame seed extract, preferably the extract of water, ethanol or a mixed solvent of the sesame seed, more preferably an extract of the solid having the extraction solvent removed. A fraction of the hexane layer obtained by fractionation with water and hexane, or an extract obtained by suspending the extract in water and then sequentially fractionating by adding hexane, ethyl acetate and butanol. By "sequentially fractionating" here is meant that fractions of the water layer continue to be used after fractionation with the solvents in the order listed above.

In addition, in the present specification, "ethyl acetate fraction of sesame seed extract" may be defined the same as "hexane fraction of sesame seed extract" except that the fractionation solvent is ethyl acetate.

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 dietary composition comprising the kohlrabi extract, hexane fraction thereof, or ethyl acetate fraction as an active ingredient.

In yet another aspect, the present invention relates to a hyperlipidemic improver composition comprising a kohlrabi extract, hexane fraction thereof, or ethyl acetate fraction 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. More specifically, it refers to a condition in which lipid components such as triglycerides, LDL cholesterol, phospholipids, and free fatty acids are increased in the blood.

In the diet composition and the hyperlipidemic composition of the present invention, the meanings of the sesame seed extract, the hexane fraction and the ethyl acetate fraction, the effective amounts thereof, and the like, as described above with respect to the obesity improver composition of the present invention are effective as they are. .

In yet another aspect, the present invention relates to a fatty liver improver composition comprising a kohlrabi extract, hexane fraction thereof, or ethyl acetate fraction as an active ingredient.

In the following Examples and Experimental Examples, in addition to the 3T3-L1 precursor adipocytes, the present inventors examined the effects of induction of fat accumulation on human liver cancer cell line HepG2, which has metabolic function as hepatocytes, and then treated samples to effect AMPK signal transduction. As in the case of treatment with 3T3-L1 progenitor cells, LBK1 (AMPK-Kinase) was activated to activate AMPK, inhibit the activity of acetyl-CoA carboxylase (ACC), and CPT-1α (carnitine palmitoyl). activating transferase-1α, inhibiting the expression of SREBP-1c (sterol regulatory element binding protein-lc) and inhibiting the expression of FASN (fatty acid synthetase), a protein in its downstream pathway. Sikkim could be confirmed. Activation of SREBP-1c is known to promote the expression of FASN, ACC and the like to promote the synthesis of triglycerides in the liver (Proc. Natl. Acad. Sci. USA, 96: 12737-12742, 1999; J. Biol Chem. 274: 30028-30032, 1999).

The results of the experiment can be said that the sesame seed extract can also be usefully used 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, the meanings of the sesame seed extract, the hexane fraction and the ethyl acetate fraction, the effective amounts thereof, and the like are effective as described above with respect to the obesity improver composition of the present invention.

 In another aspect, the present invention relates to an insulin resistance improving agent composition comprising a sesame seed extract, a hexane fraction thereof, or an ethyl acetate fraction 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, and the former is AMPK (AMP-dependent protein kinase), and the latter is TNF-α, lL-6, and resistin. ), And the like.

Since AMPK activity is accompanied by insulin resistance and hyperglycemia, studies have shown that 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).

Fatty acid synthase (FASN) and adipoocyte fatty acid-binding protein 2 (aP2), which are expressed in adipocytes, are also known to cause insulin resistance.

FASN is initially expressed in the differentiation of adipocytes (J. Biol. Chem., 255: 4745-4750 (1980)) and palmitate from acetyl-CoA and malonyl-CoA. palmitate), an FASN that stores excess energy in the form of triglycerides to cause obesity, while palmitate, a triglyceride produced by it, breaks down the pancreatic β cells and causes insulin resistance. (Proc. Natl. Acad. Sci. 95: 2498-2502 (1998)), aP2 is a potent target protein in the development of insulin resistance therapeutics, resulting in a low incidence of insulin resistance in mice lacking the gene and ap2 inhibitors reducing insulin resistance. (Nature 447: 959-965 (2007)).

The following Examples and Experimental Examples show that the E. coli extract as an active ingredient of the obesity improver composition of the present invention activates AMPK and inhibits the expression of FASN and aP2. These experimental results can be said to be useful as not only as an obesity improver but also as 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 improver composition of the present invention, the meaning of sesame seed extract, hexane fraction and ethyl acetate fraction, meaning of the effective amount thereof, and the like as described above with respect to the obesity improver composition of the present invention is effective as it is.

The obesity improver composition, the composition for diet, the hyperlipidemic improver composition, the fatty liver improver composition, and the insulin resistance improver composition (hereinafter, the "composition of the present invention") of the present invention can be understood as food compositions in specific embodiments.

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 include sweeteners, flavoring agents, bioactive ingredients, minerals, etc. in addition to the active ingredients.

Sweeteners may be used in amounts that give the food a suitable sweet taste, 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.

Flavoring agents can be used to enhance the taste or aroma, 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. In some cases, synthetic flavoring agents may be used, and synthetic flavoring agents may include esters, alcohols, aldehydes, terpenes, and the like.

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

In addition, the food composition of the present invention may contain a preservative, an emulsifier, an acidulant, a thickener, and the like, in addition to the sweetener.

Such preservatives, emulsifiers and the like are preferably added and used in very small amounts as long as the use to which they are added can be achieved. By trace amount is meant numerically expressed in the range of 0.0005% to about 0.5% by weight based on the total weight of the food composition.

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

Emulsifiers that can be used include acacia gum, carboxymethylcellulose, xanthan gum, pectin and the like.

Examples of acidulants that may be used include lead acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, and the like. Such acidulant may be added so that the food composition is at an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to the purpose of enhancing taste.

Thickeners that can be used include suspending implements, sedimenters, gel formers, swelling 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 the same as described above with respect to the extract of the kohlrabi. Such extracts may be obtained by mixing the extraction objects.

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

The pharmaceutical composition of the present invention includes oral formulations (tablets, suspensions, granules, emulsions, capsules, syrups, etc.), parenteral formulations (sterile injectable aqueous or Oily suspensions), topical formulations (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.

Excipients may be selected and used according to the formulation of the pharmaceutical composition of the present invention, for example, when the pharmaceutical composition of the present invention is prepared with an aqueous suspending agent, suitable excipients are sodium carboxymethyl cellulose, methyl cellulose, hydropropylmethylcellulose Suspending agents and dispersing agents such as sodium alginate and polyvinylpyrrolidone 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 dosage of the pharmaceutical composition of the present invention is usually 0.001 ~ 150 mg / kg body weight range, it can be administered once or divided into several times. However, since the dosage of the pharmaceutical composition of the present invention is determined in view of various related factors such as the route of administration, the age, sex, weight of the patient, and the severity of the patient, the dosage may limit the scope of the present invention in any aspect. It should not be understood as.

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 is a result of treating the ethanol extract (GE) of the cobs in 3T3-L1 of the fat precursor cells and observe the content of fat droplets and neutral fat.
Figure 2 is a result of treating the ethanol extract (GE) of the cobs in 3T3-L1 of the fat precursor cells and observe the expression level of differentiation-related proteins.
Figure 3 is a result of treating the hexane fraction (GHFr) of the ethanol extract of Kompanzees to 3T3-L1 of fat precursor cells and observe the content of fat droplets and triglycerides.
Figure 4 is a result of treating the ethyl acetate fraction (GEFr) of ethanol extract of Kokko to 3T3-L1 of the fat precursor cells and observe the content of fat droplets and neutral fat.
5 is a result of treating the hexane fraction (GHFr) and ethyl acetate fraction (GEFr) of the ethanol extract of Kokko to 3T3-L1 of fat precursor cells and observe the expression level of differentiation-related proteins.
Figure 6 is a result of treating the ethyl acetate fraction (GEFr) of ethanol extracts of Kompanzees in 3T3-L1 of adipocytes and observed the degree of phosphorylation (activation or inactivation) of AMPK and its substrate protein ACC.
Figure 7 is a result of treating the ethyl acetate fraction (GEFr) of ethanol extracts of Kompanzees to 3T3-L1 of adipose progenitor cells and observed the expression level of LKB1 and the level of ROS generation, a higher regulatory protein of AMPK.
8 is a result of treating the ethyl acetate fraction (GEFr) of the ethanol extract of Kompanzees to 3T3-L1 of the fat precursor cells and observed the expression level of UCP2 and CPT-1α involved in fatty acid oxidation.
9 is a result of treating the ethyl acetate fraction (GEFr) of the ethanol extract of the Kokko to 3T3-L1 of the progenitor cells and observed the degree of sugar absorption and lipolysis.
FIG. 10 is a ethanol extract (GE), hexane fraction (GHFr) and ethyl acetate fraction (GEFr) of the cobs, treated with HepG2, a human liver cell line, and the degree of phosphorylation (activity or Degree of inactivation).
FIG. 11 shows the ethanol extract (GE), hexane fraction (GHFr) and ethyl acetate fraction (GEFr) of the cobs, treated with HepG2, a human hepatocyte cell line, and the degree of phosphorylation (activity or Degree of inactivation).
12 is a result of treating the ethanol extract (GE), its hexane fraction (GHFr) and ethyl acetate fraction (GEFr) of the cobs in HepG2, a human liver cell line, and observed the expression level of CPT-1α.
FIG. 13 shows that ethanol extract (GE), hexane fraction (GHFr), and ethyl acetate fraction (GEFr) of the cobs are treated with HepG2, a human hepatocyte cell line, and phosphorylation degree (activation degree) of AMPK and its higher regulatory protein LKB1. Observed.
14 is a result of treating the ethanol extract (GE) of the cockle to HepG2, a human liver cell line, and observed the degree of phosphorylation (activation degree) and the expression level of SREBP-1c of AMPK.
15 is a result of treating the ethanol extract (GE) of the cockle to HepG2 and observing the expression level of FASN.

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 > Cocktail  Extract and Fraction  Produce

Cockletails were collected in the intertidal zone of eastern Jeju Island in March 2010. The collected sample was washed with tap water to remove salt, dried in the shade, and then ground to obtain a powder. In order to obtain the ethanol extract of the sample, 200 g of the powder was extracted in 2 L of 80% ethanol for 48 hours and filtered under reduced pressure. The remaining residue from the filtration was extracted twice with the addition of the same amount of 80% ethanol, and then concentrated with a primary rotary extractor (Buchi, R-200, Switzerland). Ethanol extract obtained after removing ethanol from the concentrate was lyophilized and used for fractionation or cell experiment. The solvent fraction of the ethanol extract was obtained by suspending the extract by adding water to the extract, and then performing hexane, ethyl acetate, butanol, and water three times in succession, followed by concentration under reduced pressure and lyophilization. 80% ethanol extract (GE), hexane fractionation layer (GHFr), ethyl acetate fractionation layer (GEFr) was dissolved in DMSO, butanol and water fractionation layer in PBS (phosphate buffered saline) was used for cell experiments.

< Experimental Example > Cocktail  Extract and Fraction  Obesity improving activity, fatty liver improvement activity, hyperlipidemia improving activity and insulin resistance improving activity experiment

One. Experimental Method

<1-1> Cell Culture

Mouse-derived 3T3-L1 progenitor cell lines were purchased from the American Type Culture Collection (ATCC). Cell culture was performed using Dulbecco's modified Eagle's medium (DMEM; Gibco, USA) supplemented with 10% bovine calf serum (BCS; Gibco, USA) and 1% penicillin / streptomysin (P / S; Gibco, USA). % CO ₂ It was performed under conditions. Cells were passaged when 70% of the cells were filled at the bottom of the T-75 cell culture flask.

HepG2 cell lines were purchased from the American Type Culture Collection (ATCC). Cell cultures were performed using Dulbecco's modified Eagle's medium (DMEM; Gibco, USA) medium supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin / streptomysin (P / S; Gibco, USA). 5% CO ₂ It was performed in an incubator under conditions (Sanyo, Japan). Cells were maintained in passages when the cells were 80% full at the bottom of a 100 mm cell culture dish.

<1-2> 3 T3 - L1 precursor adipocyte differentiation and sample processing

Induction of differentiation into adipocytes was performed by modifying the method of Harmon and Harp (2001). 3T3-L1 progenitor cells were inoculated into 6 well cell culture plates (1.6 or 2.0 x 10 ⁵ cells / well) using DMEM medium supplemented with 10% BCS and 1% P / S and incubated for 48 hours to pre-confluent It was made to be in a state. 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 Differentiation was induced for 2 days (day 2 of induction of differentiation) by exchange with -1-methylxanthine (IBMX; Sigma, USA) and DMEM medium containing 5 μg / ml insulin (Sigma, USA). In order to analyze the effect of E. coli ethanol extract and fraction layer on adipocyte differentiation, each sample was collected in differentiation induction (day 0) (ethanol extract: 2, 10, 50 and 100 ㎍ / ml; hexane and ethyl acetate Fractionated layers: 1, 5, 25 and 50 μg / ml) were added to the differentiation induction medium. Two days after induction of differentiation, the cell cultures were exchanged with DMEM medium containing 10% FBS, 1% P / S, and 5 μg / ml insulin. On the fourth day of induction, the cell cultures were exchanged every two days with DMEM medium containing only insulin. It was.

In order to analyze the effect of AMP-activated protein kinase (AMPK) and its substrate, acetyl Co-A carboxylase (ACC) on phosphorylation and expression of its subgenes, the differentiation of adipocytes was carried out as follows. It was. Cells in confluent (day 0 differentiation) were induced to differentiate for 2 days in DMEM medium containing 10% FBS, 1% P / S, 1 μM DEXA., 0.5 mM IBMX and 5 μg / ml insulin. After 2 days of induction of differentiation, the medium was exchanged with DMEM medium containing 10% FBS, 1% P / S and 5 μg / ml insulin, and again after 2 days (day 4 of differentiation induction) every 2 days until use in the experiment. Exchange with DMEM medium containing 10% FBS and 1% P / S. After 8-10 days of differentiation induction, the medium was removed from the culture vessel, washed twice with phosphate buffered saline (PBS), exchanged with DMEM medium, incubated for 6 hours in serum-free stavation, and then 50 ㎍ / ml. Phosphorylation of AMPK and ACC was confirmed by treatment of the ethyl acetate fractions of 12 hours with time (0, 1, 3, 6, 12 and 24 hours) or with concentration (1, 5, 25 and 50 μg / ml). .

Induction of Fat Accumulation in HepG2 Cells

Induction of fat accumulation in HepG2 cells was performed by modifying the method of Gomez-Lechon et al. (2007). HepG2 cells were inoculated into 6 well cell culture plates (1.0 × 10 ⁶ cells / well) using DMEM medium containing 10% FBS and 1% P / S and incubated for 48 hours. It was about% cold. About 80% of the cells were cold and exchanged with medium containing 200 μM of free fatty acids, oleic acid and plamitic acid, and cultured for 24 hours to induce fat accumulation in HepG2 cells.

<1-4> Oil - Red O Dyeing

Oil Red O staining and quantitative analysis were performed by Cho et al. (2003). After 7-9 days of differentiation induction cells were washed twice with PBS, fixed with 3.7% formalin [diluted with 37% formaldehyde solution (Sigma, USA) 1/10] diluted in PBS for 1 hour, and then washed twice with distilled water. It was. Oil Red O staining solution was prepared by diluting 0.6% Oil Red O (Sigma, USA) diluted with isopropanol (Merck, Germany) and distilled water 6: 4 and then filtering. The washed cells were stained with Oil Red O staining solution for 1 hour and washed three times with distilled water and observed under a microscope. In order to quantify the content of triglycerides of dyed fatty droplets, isopropanol containing 4% NP-40 (Amresco, USA) was added to dissolve Oil Red O and absorbance was measured at 520 nm with a microreader.

<1-5> Free Glycerol release measurement

Free glycerol content was measured using a free glycerol reagent kit (Sigma, USA). After 3T3-L1 precursor adipocytes were cultured and differentiated for 8 days according to the differentiation induction method, the ethyl acetate fractionation layer was treated with concentrations (0, 2, 10 and 50 μg / ml) for 48 hours. 5 μl of the culture solution was added to 0.4 ml of the free glycerol measurement reagent and reacted at 37 ° C. for 10 minutes, and the absorbance was measured at 540 nm using a micro reader. In order to quantify free glycerol, standard glycerol solution (Sigma, USA) was quantitated by reacting the same as above by making the concentrations of 0, 4.0625, 8.125, 16.25, 32.5, 65, 130 and 260 ㎍ glycerol / ml, and repeated three times. .

<1-6> RNA isolation and real - time PCR

Total RNA for real-time PCR in adipocytes was treated by concentration (2, 10 and 50 ㎍ / ml) for 48 hours to cells on day 8 of differentiation, and then the cells were collected and isolated. 500 μl of Trizol Reagent (Invitrogen, USA) was added to homogenize the cells at room temperature for 5-10 minutes, then transferred to a tube and centrifuged (12,000 × g, 15 minutes) by adding 100 μl of chloroform. 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 ratio of A260 / A280 nm in the range of 1.8-2.0 was treated with DNase (Wako, Japan) to isolate only pure RNA and used for the experiment.

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

Real-time PCR for 5 μl cDNA, each primer 0.4 μl (10 pmol / L /), 2 μl nuclease-free water and 6 μl iQ ^TM SYBR After mixing the Green Supermix (Bio-rad, USA) was performed using a Peltier thermal cycler (Bio-rad, USA) real time PCR instrument. The PCR conditions were amplified 49 times at 95 ℃ / 20 seconds, 65 ℃ / 20 seconds, 72 ℃ / 30 seconds, and the fluorescence was measured every time amplification. The reaction curve was analyzed by melting to 95 ℃. 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 are shown in [Table 1].

<Primer>

Total RNA for real time-PCR analysis in hepatocytes was inoculated into HepG2 cells in 6 well cell culture plates (1.0 × 10 ⁶ cells / well) GHFr, GEFr samples: 12.5, 25, 50 μg / mL) were incubated for 72 hours and then separated. Cells were homogenized by adding Tri Reagent (Molecular Research Center, Inc, USA), followed by centrifugation (12,000 × g, 4 ° C., 15 minutes) by adding chloroform thereto. The supernatant was recovered and centrifuged (12,000 × g, 4 ° C., 8 minutes) by adding the same amount of isopropanol to precipitate RNA. Precipitated RNA was washed with 75% ethanol, dried, dissolved in 50 μl of nuclease-free distilled water (NFDW; Amresco, USA), and then absorbed at 260 nm using a nanodrop ND 2000 spectrophotometer (Nanodrop, USA). Quantitation was performed and treated with DNase (Wako, Japan). RNA samples with values in the range A260 / A280 nm in the range 1.6-2.0 were used for the experiment.

cDNA was synthesized using Maxime RT PreMix Kit (Oligo dT primer, iNtRON Biotechnology, Korea). 1 ㎍ total RNA and NFDW in a total of 20 ㎕ in the kit and reacted for 45 minutes 60 minutes, 95 minutes 5 minutes at 95 ℃ synthesized cDNA after measuring the absorbance of 260 nm and added NFDW to the final concentration of cDNA Was adjusted to 0.25 μg / mL.

Real-time polymerase chain reaction (PCR) was performed with 2 μl of cDNA, 1 μl (10 pM / μl) of each primer, 3.5 μl of NFDW and 7.5 μl of iQ ^TM SYBR. Green Supermix (Bio-rad, USA) was mixed and then performed using a Chromo4 real time PCR (Bio-rad, USA) instrument. PCR conditions were amplified 50 times at 95 ℃ / 20 seconds, 63.5 ℃ / 20 seconds, 72 ℃ / 30 seconds and the results were measured by absorbance each time. Results were analyzed by gene expression analysis for chromo4 real-time PCR detection system v1.10 (Bio-rad, USA). The base sequence of the primer used in the real-time PCR and the size of the expected product are shown in [Table 2].

<Primer>

<1-7> Western blot

The sample treated adipocytes were washed twice with PBS, followed by 1 mM PMSF, 1 mM Na ₃ VO4, 1 mM NaF, 1 μg / ml aprotinin, 1 μg / ml pepstatin, 1 μg / ml leupeptin and 10% RIPA lysis buffer. (Upstate Biotechnology, USA) using a protein separation reagent containing for 1 hour vortexing and digested, then centrifuged (15000 × rpm, 4 ℃, 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 μg of protein was electrolyzed on 10% (PPAR, C / EBPa, phospho-AMPK, AMPKα, LKB1, phospho-LKB1 and β-actin), 6% (phospho-ACC) or 15% (aP2) SDS-polyacrylamide gel After separation by electrophoresis, polyvinylidene difluoride (PVDF) membrane (Milipore, USA) was transferred to (100 V, 120 minutes). 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 and then reacted with the primary antibody. Primary antibodies used for protein analysis were PPAR antibody (1: 500, Santa Cruz, USA), C / EBPa antibody (1: 1,000, Santa Cruz, USA), 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), LKB1 and p-LKB1 antibody (1: 1,000, Santa Cruz, USA), A- FABP antibody (aP2; 1: 5,000, Santa Cruz, USA), β-actin antibody clone AC-74 (1: 10,000, Sigma, USA) and the like was performed overnight at 4 ℃. Membrane was washed with 0.1% TTBS solution and diluted peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, USA) at 1: 2,000, 1: 5,000 or 1: 10,000 for 1 hour. 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).

HepG2 cells were inoculated into 6 well cell culture plates (1.0 × 10 ⁶ cells / well) for the analysis of wester blot in hepatocytes, and the samples were incubated hourly (30 minutes, 1 hour, 2 hours, 6 hours, 12 hours). Time; GE samples: 100 μg / mL; GHFr, GEFr samples: 50 μg / mL) and by concentration (GE samples: 25, 50, 100 μg / mL; GHFr, GEFr samples: 12.5, 25, 50 μg / mL, 12 hours incubation). Cells were then washed twice with cold phosphate buffered saline (PBS) followed by 10% RIPA lysis buffer (Upstate Biotechnology, USA) and 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM Na ₃ VO ₄ , 1 mM NaF, 1 μg / Supernatants were obtained by digestion for 1 hour with protein separation reagent containing mL aprotinin, 1 μg / mL pepstatin, and 1 μg / mL leupeptin, followed by centrifugation (15,000 rpm, 4, 20 minutes). Proteins were measured and absorbed at 595 nm with a micro reader using a Bio-rad protein assay kit (Bio-rad, USA). Protein (40 μg / μl) was electrophoresis (100 V, 120 min) on 8% SDS-polyacrylamide gel and then transferred to poly vinylidene difluoride (PVDF) membrane (Milipore, USA) (100 V, 90 min). . The PVDF membrane with protein transfer was blocked with 5% skim milk (BD, USA) for 1 hour at room temperature, and then reacted with the primary antibody. The primary antibody reaction was AMP-activated protein kinase a (AMPKα) (23A3) antibody (1: 1,000; Cell Signaling, USA), phospho-AMPKα (Thr 172) (40H9) antibody (1: 1,000; Cell Signaling, USA) , phospho-acetyl-CoA carboxylase (ACC) (Ser 79) antibody (1: 1,000; Cell Signaling, USA), LKB1 (T-22) antibody (1: 1,000; Santa Cruz, USA), phospho-LKB1 (Ser 431 ) -R antibody (1: 1,000; Santa Cruz, USA), sterol regulatory element-binding protein 1c (SREBP-1c) antibody (1: 1,000; BD, USA) was reacted at 4 ° C. overnight, β-actin clone AC -74 antibody (1: 10,000; Sigma, USA) was performed at room temperature for 1 hour. After completion of the primary antibody reaction, PVDF membrane was washed 5 times with Tris buffered saline (TTBS) solution containing 0.1% Tween 20. As a secondary antibody, anti-mouse or anti-rabbit IgG (Jackson ImmunoResearch, USA) conjugated with horse radish peroxidase (HRP) was diluted to 1: 5,000 or 1: 10,000 for 1 hour and then washed 5 times with TTBS. It was. The expression level of each protein was detected by X-ray film (Agfa Gevaert NV, Belgium) by reacting with a WEST-ZOL western blot detection system (iNtRON Biotechology, Korea).

<1-8> Glucose uptake analysis

To determine the effect of the sample on glucose uptake into adipocytes, glucose uptake analysis was performed using 2-Deoxy-D glucose (2-DOG) labeled with ³ H. 3T3-L1 adipocytes were induced in differentiation in 12 well culture plates as described above and used for experiments on days 8-10. Differentiated adipocytes were washed twice with serum-free medium without serum, and then 1 mL of the same medium was added and cultured at 37 ° C. for 2-6 hours. The cultured cells were washed twice with Krebs-Ringer-Hepes (KRH) buffer [20 mM Hepes, 136 mM NaCl, 4.7 mM KCl, 1.25 mM MgSO ₄ , 1.25 mM CaCl ₂ , (pH 7.4)] to remove the medium. Incubate for 1 hour by adding the samples dissolved in the same buffer (2, 10 and 50 ㎍ / ㎖), and then treated with 100 nM diluted in 100 μl KRH buffer and incubated for 20 minutes, 37 MBq 2-deoxy- A mixture of D- [ ³ H] -glucose (Amersham Biosciences, Sweden) and 1 mM 2-Deoxy-D-glucose (Sigma, USA) was added to 100 μl with a final concentration of 3.7 MBq and 0.1 mM, respectively, for 20 minutes. Incubated. After removing the culture solution and washing three times with cold PBS, 1 ml of 1% Triton X-100 lysis buffer was added to measure the amount of 2-DOG absorbed into the cells, followed by incubation at 37 ° C. for about 10 minutes. Radioactivity within was measured using a Liquid Scintillation Counter (Tri-Carb 2700TR Packard, Meriden, CT, USA).

<1-9> Intracellular ROS Measurement

Intracellular reactive oxygen species was identified as 5- (and-6) -chloromethyl-2 ', 7'-dichlorodihydrofluorecein diacetate acetyl ester (CM-H2DCFDA; Molecular Probes, Eugene, OR, USA, according to the method of Hwang et al. (2005, 2007). ROS staining was confirmed. Differentiation-induced 3T3-L1 adipocytes were fasted in DMEM medium containing 0.1% BSA for 6 hours and diluted in DMEM (GEFr, N- acetylcystein) at different concentrations (GEFr; 0, 10 and 50 μg / Ml, N- acetylcystein; 5 mM) for 12 hours. After the lapse of time, the cells were washed twice with PBS and fixed with 3.7% formaldhehyde for 15 minutes.Then, CM-H2DCFDA was added to the final concentration of 10 μM per well and stained at room temperature for 30 minutes with light blocking. It was confirmed using.

<1-10> Statistical processing

The experimental results were expressed as mean ± standard deviation and tested for significance by student's t-test.

2. Experiment result

<2-1> Inhibitory Effects of Ethanol Extracts and Their Fractions from Induction of Profat Cells

First, MTT assay was performed to determine the cytotoxicity of 3T3-L1 precursor adipocytes of the E. coli ethanol extract (GE). The cell experiment was performed at the highest concentration of 100 ㎍ / ml which did not affect cytotoxicity. .

To investigate the effect of GE on the differentiation of 3T3-L1 progenitor cells, differentiation-inducing substance and GE (2, 10, 50 and 100 ㎍ / ml) were treated simultaneously with differentiation-inducing oil Red O staining. Fat droplets were observed. As shown in FIG. 1A, it was confirmed that the fat droplets were reduced in a concentration-dependent manner in the experimental group treated with GE. As a result of quantifying the Oil Red O staining intensity, the experimental group treated with GE at 2, 10, 50 and 100 μg / ml concentrations was 106.9 ± 19.4, 91.3 ± 18.5 compared to the negative control (100 ± 18.0%). , 15.6 ± 2.8 and 13.7 ± 0.6% (Fig. 1B). GE significantly inhibited the differentiation of 3T3-L1 profat cells from a concentration of 50 μg / ml (FIG. 1B). The expression of adipocyte differentiation-related proteins was analyzed by Western blot in order to confirm the effect of morphological lipogenic formation by GE at the molecular level. Peroxisome proliferator activated receptor γ (PPARγ), CCAAT / enhancer binding protein α (C / EBPα), a key transcription factor that regulates adipocyte differentiation, and fatty acid binding protein (aP2) ) Was found to decrease in concentration dependently compared to the control (Fig. 2).

In order to identify the fractions containing GE's pro-dipotent cell differentiation-inhibiting active ingredients, GE fractions were solvent fractionated and the cytotoxicity of each fraction was confirmed by MTT assay. The experiment was conducted.

In addition, the hexane fraction (GHFr) and ethyl acetate fraction (GEFr) were treated at concentrations of 1, 5, 25, and 50 ㎍ / ml, respectively, with the induction of 3T3-L1 progenitor cell differentiation. In the experimental group treated with GHFr, a decrease in fat droplets was confirmed from the lowest concentration of 1 μg / ml, and it was confirmed that the concentration of fat droplets was effectively inhibited at a concentration higher than 5 μg / ml (FIG. 3A). In addition, as shown in the results of Fig. 3B quantitatively analyzing the fat droplets from the group treated with GHFr 1 ㎍ / ㎖ showed a significant fat droplet reduction effect compared to the control group.

In the experimental group treated with GEFr, the formation of fat droplets was significantly inhibited from the concentration of 1 μg / ml compared with the control group (FIG. 4A). In the quantitative analysis, it was confirmed that all treatment groups significantly inhibited the generation of fatty droplets compared to the control group (FIG. 4B). As a result of confirming the fat accumulation inhibitory activity of GHFr and GEFr at PPAR, C / EBPa and aP2 protein expression level, GHFr was treated at a concentration of 5 ㎍ / ml for the GHFr (Fig. 5A). It was confirmed that these protein expression was significantly reduced in all one group (Fig. 5B).

<2-2> 3 T3 - L1 kkomul kkosiraegi ethyl acetate layer fraction (GEFr) in adipocytes is

In differentiated fat cells AMPK  Influence on signal transmission

<2-2-1> AMPK Phosphorylation Inducing Activity

In the previous experiments, it was confirmed that the ethyl acetate fraction layer (GEFr) had the best inhibitory effect on adipocyte differentiation among the solvent fractions of the kohlrabi. We investigated the effect of GEFr on AMP-activated protein kinase (AMPK) signaling. First, the fully differentiated adipocytes were treated with GEFr (50 μg / ml) and analyzed for AMPK phosphorylation by time (1, 3, 6, 12 and 24 hours). After 12 minutes after treatment with GEFr in adipocytes, it was confirmed that AMPK and its substrate, acetyl-CoA carboxylase (ACC), were phosphorylated (FIG. 6A). In addition, it was confirmed that the AMPK phosphorylation increased in a concentration-dependent manner when the cells were treated with GEFr for 12 hours at different concentrations (0, 1, 5, 25 and 50 μg / ml) (FIG. 6B). However, phosphorylation of ACC with phosphorylation of AMPK also increased with increasing concentration from 5 μg / ml treatment group, but did not increase at higher concentrations (25 and 50 μg / ml) (FIG. 6B).

<2-2-2> LKB1 Phosphorylation and ROS Generation

 Since activation of AMPK is known to be achieved by phosphorylation of the residue of Thr172 position of a-subunit of AMPK by LKB1, also known as upstream AMPK-Kinase, the phosphorylation of LKB1 was confirmed by Western blot. Treatment of differentiation-induced adipocytes with concentrations of 0, 1, 5, 25 and 50 ㎍ / ml, respectively, showed that LKB1 phosphorylation was significantly increased from the concentration of 1 ㎍ / ml (Fig. 7A). ).

In addition, reactive oxygen species (ROS) is known to be a factor that activates AMPK by acting as oxidative stress in the cell, so the effect of GEFr on ROS production was confirmed. ROS generated when 10, 50 μg / ml of GEFr were treated to differentiated adipocytes through DCFH-DA fluorescence staining, showed a concentration-dependent increase in ROS (FIG. 7B). Treatment with N- acetylcysteine ( NAC ), known as an antioxidant, to confirm whether the production of ROS is effected by treatment with GEFr, resulted in a decrease in the production of ROS (FIG. 7B).

<2-2-3> Expression of fatty acid β-oxidation related genes

Since AMPK activation is known to promote fatty acid β-oxidation, expression of fatty acid β-oxidation related genes (CPT-1α, UCP2 ) following GEFr treatment was analyzed by real-time PCR. Carnitine palmitoyltransferase 1α (CPT-1α) and uncoupling protein 2 In the case of (UCP2), the expression of mRNA was increased in the group treated with GEFr at a concentration of 50 μg / ml compared to the group treated with DMSO as a control (FIGS. 8A and B).

<2-2-4> Sugar absorption ( glucose uptake ) and lipolysis effects

Glucose uptake experiment using 2-deoxy-D-glucose labeled with ³ H in adipocytes confirmed the effect of GEFr on glucose uptake. GEFr was applied to differentiated adipocytes at concentrations of 2, 10 and 50 μg / ml, respectively. It was confirmed that no effect on the absorption (Fig. 9A). However, GEFr was found to have a lipolytic activity that increases the concentration of glycerol from the adipocytes to the medium (Fig. 9B).

<2-3> Effects of Sesame Seed Extract and Fractions on AMPK Signaling in HepG2 Cells Induced Fat Accumulation

MTT analysis in HepG2 cells 100 μg / ml for ethanol extract (GE) and 50 ㎍ / ml for the nucleic acid fraction (GHFr) and ethyl acetate fraction (GEFr) at the non-cytotoxic concentration through LDH analysis. Cell experiments were conducted by treatment. In order to analyze the effects of GE, GHFr, and GEFr on fatty acidization, HepG2 cells inducing fat accumulation were used. The cells were treated with oleic acid and palmitic acid and incubated for 24 hours to induce fat accumulation. Then, the samples were processed and cultured and the phosphorylation of AMPK and its substrate, ACC, was confirmed by Western blot. Fat-induced HepG2 cells were treated with a constant concentration sample (GE, 100 μg / ml; GHFr, 50 μg / ml; GEFr, 50 μg / ml) to analyze the phosphorylation of AMPK and ACC over time (FIG. 10). . The activation of AMPK was confirmed to start from 2 hours after sample treatment and increase to 12 hours. Phosphorylation of ACC, a substrate of AMPK, was highest at 2 hours after treatment with AMPK-activated samples and decreased over time.

Since each sample was treated for each concentration to determine the effect of the concentration of the sample. Each sample was treated for each concentration, and after culturing for 12 hours for p-AMPKα and for 2 hours for p-ACC, the protein was separated and the amount of protein was confirmed. As a result, both p-ACC and p-AMPKα increased the amount of protein in a concentration-dependent manner for each sample (Fig. 11). Based on the results, the mRNA expression level of CPT-1α expressed in the liver was confirmed by real-time PCR to confirm the activation of CPT1, a sub-path of AMPK. As a result, it was confirmed that the expression level of CPT-1α mRNA increased significantly in a concentration-dependent manner (FIG. 12).

In addition, the phosphorylation of LKB1, the upper kinase, was confirmed by searching the upper pathway of AMPK to find out which pathway is activated by AMPK. In the case of LKB1 there was no difference in the amount of expression, but in the case of p-LKB1, the amount of expression increased in a concentration-dependent manner (FIG. 13).

In order to determine the fatty acid synthesis inhibitory effect using GE samples, the protein amount of sterol regulatory element binding protein 1c (SREBP-1c) was confirmed, and the amount of SREBP-1c expression was reduced (FIG. 14). In addition, as a result of real-time PCR confirming the expression level of mRAN of the lower pathway fatty acid synthease (FASN), the expression level was significantly decreased in a concentration-dependent manner (Fig. 15).

<110> Industry-Academic Cooperation Foundation Jeju National University <120> Composition for Improving Obesity and Fatty Liver <130> PP10-000-Jeju <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 accctgaggc atctattgac a 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tgacatactc ccacagatgg c 21 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ggtcggagat accagagcac 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tgaggttggc tttcaggaga 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 aggctgtgct gtccctgtat 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 acccaagaag gaaggctgga 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 cactacaagg acatgggcaa g 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 caacttcagc ctctgttcca c 21 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 gggacaacct ggagttcttc 20 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 cacttgatga gtggggagat g 21 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 cactcttcca gccttccttc 20 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 gtgatctcct tctgcatcct g 21

Claims (18)

Obesity improver composition comprising sesame seed extract, hexane fraction thereof, or ethyl acetate fraction 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.
Hyperlipidemic improver composition comprising sesame seed extract, hexane fraction thereof, or ethyl acetate fraction 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 improver composition comprising sesame seed extract, hexane fraction thereof, or ethyl acetate fraction 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 improver composition comprising a kohlrabi extract, hexane fraction thereof, or ethyl acetate fraction 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.
Dietary food composition comprising the extract of Kok Kok Seol, hexane fraction thereof, or ethyl acetate fraction as an active ingredient.
18. The method of claim 17,
The extract is a dietary food composition, characterized in that obtained by using water, ethanol or a mixed solvent thereof

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