KR101542694B1 - Composition comprising water extract of hulled barley for preventing and treating obesity - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising a water extract of hulled barley for preventing or treating obesity. The water extract of hulled barley of the present invention is confirmed to have an adipocyte differentiation and suppression effect. Therefore, the invention is expected to be used in a target treatment for preventing or treating obesity in a more fundamental manner.

Description

[0001] The present invention relates to a composition for preventing or treating obesity,

The present invention relates to a composition for the prevention or treatment of obesity, which comprises a water extract of a barnacle (water) as an active ingredient. More specifically, the present invention relates to a pharmaceutical composition for preventing or treating obesity through inhibition of adipocyte differentiation.

Obesity is caused by abnormal enlargement of subcutaneous fat tissue caused by accumulation of excess energy in the body when the metabolic process imbalance occurs due to endocrine factors, genetic factors, and socio - environmental factors. The hypertrophy of adipose tissue is a phenomenon that the size of adipocyte increases (adipocyte hypertrophy) and the number of adipocyte hyperplasia increases (adipocyte hyperplasia), which also affects the localization of the venous-lymphatic system, . Thus, obesity is perceived as an independent disease in itself, and the World Health Organization treats obesity as a global nutritional problem and recognizes it as a disease to be treated rather than a simple risk factor that damages health.

In this situation, obesity population is increasing in modern society. Indeed, in the United States, adults have doubled in size over the past two decades, and in 2008, 27% of adults were overweight and 60% were overweight. Children and adolescents are even more serious, tripling over the past 30 years. Therefore, about 6% of health expenditure is paid for obesity treatment, and the domestic obesity treatment market is close to 1 trillion won. In addition, obesity treatments are ranked as a representative QOL (quality of life) improvement along with erectile dysfunction treatment and alopecia treatment.

Current therapeutic agents for obesity can be categorized as medications that affect the appetite by acting on the central nervous system, and drugs that act on the gastrointestinal tract and inhibit absorption, depending on the functional group. Drugs acting on the central nervous system include fenfluramine and dexfenfluramine, which inhibit serotonin (5-HT) reuptake, ephedrine and caffeine, which inhibit noradrenaline reuptake, and sibutramine, which is recently co-acting on serotonin and noradrenergic nervous system have. The drug that acts on the gastrointestinal tract and inhibits obesity is orlistat, which is approved as a treatment for obesity by inhibiting the lipase produced by the pancreas and reducing the absorption of fat. However, among the drugs used, fenfluramine, an appetite suppressant, was classified as a drug substance and was prohibited from causing side effects such as primary pulmonary hypertension or heart valve lesion. Other appetite suppressing drugs also caused blood pressure reduction or lactic acidosis And it is limited to patients with heart failure and kidney disease. Orlistat, an inhibitor of lipolysis, causes gastrointestinal side effects, causing discomfort and inhibiting the absorption of fat-soluble vitamins. Sibutramine, approved by the US FDA for long-term obesity treatment, causes cardiovascular diseases such as hypertrophy or dizziness, Was prohibited.

One of the proposals as an alternative for preventing and inhibiting the side effects of the above drugs and more fundamental obesity is an adipocyte differentiation inhibitor. Until the early 1990s, adipose tissue was mainly composed of adipocytes and was considered only as an energy reservoir to store the energy needed for the human body in the form of triglycerides and to decompose them if necessary. However, since the mid - 1990s, it has been found that adipose tissue functions as an endocrine organ that secretes substances that can affect other tissues, rather than just energy stores. Currently, there are TNF-α, IL-6, IL-8, plasminogen activator inhibitor-1, angiotensin-II, leptin and adiponectin, which are found in various metabolic diseases and cardiovascular diseases It is known to cause disease. Energy storage and endocrine secretion in adipose tissue are closely related to the differentiation and growth of adipocytes and occur simultaneously with lipogenesis. The growth of adipocytes is a process in which non-large adipocytes are formed by accumulation of triglyceride in differentiated adipocytes without changing the number of adipocytes. The obesity due to the increase in non-adipocyte cells is frequently observed in adults and regulated by diet It is possible. On the other hand, obesity due to adipocyte differentiation is mainly found in pediatric or obesity obesity, and it is important to block the differentiation of adipose precursor cells into adipocytes because it is difficult to control by diet. Accordingly, studies have been conducted on an obesity treatment drug capable of regulating the number of adipocytes produced by inhibiting the differentiation of adipocytes and regulating the extra energy accumulated therefrom (Korean Patent Laid-Open No. 10-2012-0001100) However,

Disclosure of the Invention The present invention has been made in order to solve the above problems, and the present inventors have confirmed the efficacy of the extract of the bovine corn borer on inhibition of adipocyte differentiation and accomplished the present invention on the basis thereof.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating obesity, which comprises an extract of T. obovata as an active ingredient.

Another object of the present invention is to provide a health functional food composition for preventing or ameliorating obesity, which comprises an extract of T. obovata as an active ingredient.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating obesity, which comprises an extract of T. obovata as an active ingredient.

In one embodiment of the present invention, the obesity is characterized by a hormone deficiency due to a high fat diet or menopause.

In another embodiment of the present invention, the composition is characterized by inhibiting adipocyte differentiation.

In another embodiment of the present invention, the composition is characterized in that mRNA expression of one or more genes selected from the group consisting of PPAR gamma, aP2, C / EBPa, and CD36 is reduced.

The present invention provides a health functional food composition for preventing or ameliorating obesity, which comprises an extract of T. obovata as an active ingredient.

In one embodiment of the present invention, the obesity is characterized by a hormone deficiency due to a high fat diet or menopause.

The present invention provides a method of treating obesity comprising the step of administering the pharmaceutical composition to a subject.

The present invention provides a therapeutic use of a composition comprising a bovine serum extract for the treatment of obesity.

The composition according to the present invention contains an extract of T. oblongum as an active ingredient and the inhibition of adipocyte differentiation and weight loss by the extract of T. obtusifolia can be usefully used as a pharmaceutical composition for preventing or treating obesity do.

FIG. 1 is a schematic diagram showing a method for preparing a curd extract and a barley extract according to a heat treatment temperature and an extraction solvent.
Fig. 2 shows the result of 8 days of treatment with 8 Reduced Ovary extracts in C3H10T1 / 2 cell line for 8 days in an adipocyte differentiation medium and staining with Oil Red O. Fig.
FIG. 3 shows the result of 8 days of treatment of eight barbie extracts in C3H10T1 / 2 cell line for 8 days in an adipocyte differentiation medium, followed by staining with Oil Red O. FIG.
FIG. 4 shows the results of treatment of AHB210 with heat-treated at 210 ° C in a 3T3-L1 cell line and treated with distilled water for 8 days in an adipocyte differentiation medium, followed by staining with Oil Red O. FIG.
FIG. 5 shows the result of MTT assay for the AHB210 extract of Bacillus anthracis, which was heat-treated at 210 ° C for 20 minutes in a 3T3-L1 cell line.
FIG. 6 shows the results of the real-time PCR of PPARγ, aP2, and C / EBPα (FIG. 6) associated with adipocyte differentiation after 8 days of treatment with AHB210 in a C3H10T1 / 2 cell line at 210 ° C. for 20 minutes , And the expression level of mRNA of CD36.
FIG. 7 shows the results of body weight change after administration of AHB 210 in a high-fat diet-induced C57BL / 6 mice treated with high fat diet at 210 ° C for 20 minutes (ND; Control group (n = 5), HFD, control group (n = 5), HFD + AHB15, high fat diet and 15 mg / kg daily dose (n = 4), HFD + AHB50 (n = 4) treated with the high-fat diet and 50 mg / kg daily dose of the bovine extract.
FIG. 8 shows the results of a comparison of the total dietary intake of obese-induced C57BL / 6 mice with high-fat diet.
FIG. 9 shows the results of a comparison of organ weights such as fat pad, liver, spleen and kidney in obese-induced C57BL / 6 mice administered with a high fat diet.
FIG. 10 shows the result of comparing the fat and liver tissues with H & E (hematoxylin and eosin) staining in obesity-induced C57BL / 6 mice administered with a high fat diet.
FIG. 11 shows changes in glucose, triglyceride, fatty acid, LDL, cholesterol, etc. in the blood of the HFD + bovine group in the obese-induced C57BL / 6 mice administered with the high fat diet.
FIG. 12 shows the results of testing insulin sensitivity of HFD + bovine group in obesity-induced C57BL / 6 mice by high-fat diet.
Figure 13 is a graph showing changes in body weight after 8 weeks in an 8-week ovariectomy (OVX) animal experiment using SD rats (Sham: a control group without ovariectomy (n = 5 (N = 5), OVX + AHB15 (n = 4), orally administered with 15 mg / kg of bovine extract after ovariectomy.

In the present invention, inhibition of adipocyte differentiation of C3T10T1 / 2 cells, stem cells derived from 3T3-L1 cells and fibroblasts derived from lipid precursor cells by water extracts, and expression of PPARγ, aP2, C / EBPα, and CD36 mRNA And decreased expression. In addition, the inventors of the present invention completed the present invention on the basis of confirming the weight loss by the extract of the burdock water in the obesity induced by the high fat diet or the hormone deficiency.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating obesity, which comprises an extract of T. obovata as an active ingredient.

As used herein, the term "prophylactic " means any action that inhibits obesity or delays onset by administration of a pharmaceutical composition according to the present invention.

The term "treatment" as used in the present invention means all the actions that are improved by the administration of the pharmaceutical composition according to the present invention, but are changed in a favorable manner.

As used in the present invention, the term "burden" distinguishes it from a readily falling seed barb, which is one of the types of barley in which the inner and outer spirals closely adhere to the grain particles after maturity. In other words, barley is a plant belonging to the genus of barbarians and rice bran. It is an annual or biennial plant. It is divided into two types, ie, "barley" and "barley", depending on whether the fruit husk attaches to the seed or falls off easily. . The weight of 1 liter is about 600 ~ 700g of burdock, 800g of barley, and its specific gravity is 1.1, and the barley is 1.2. Barley is good in cold resistance and is cultivated mostly in the neck, while barley is generally grown in the southern part of Daejeon because it is weaker than cover-berry. It is cultivated mainly in Hunan area, . In the present invention, an extract is obtained by using a coverstock having the above-mentioned characteristics, and a pharmaceutical composition containing the extract as an active ingredient is provided.

The term "obesity" which is a preventive and therapeutic disease caused by the composition of the present invention means a state in which the fat cells multiply and differentiate in the body due to metabolic disorders and the fat is accumulated excessively, In the case of relative increase, the number and volume of adipocytes are increased, resulting in an increase in the mass of adipose tissue. Obesity at the cellular level means an increase in the number and volume of adipocytes due to promotion of proliferation and differentiation of adipocytes.

In the present invention, obesity can be caused by hormone deficiency due to high fat diet or menopause, but is not limited thereto.

In the present invention, the bovine water extract can be extracted according to a conventional method known in the art for extracting an extract from a natural product, that is, under ordinary temperature and pressure conditions. Examples of the extraction method include hot water extraction, , Reflux extraction, ultrasonic extraction and the like, but it can be extracted preferably using distilled water as a solvent after heat treatment at 100 ° C to 210 ° C.

The bovine extract, which is an active ingredient contained in the composition of the present invention, inhibits adipocyte differentiation and reduces the expression of PPARγ, aP2, C / EBPα, and CD36 mRNA, and can be used for improving, preventing or treating obesity .

Adipocyte differentiation or adipogenesis refers to the process of accumulation of triglyceride by proliferation of adipose precursor cells and differentiation into mature adipocytes and is a risk factor for diseases such as obesity, diabetes, fatty liver and heart disease It is known. Adipocytes are differentiated from multipotent mesenchymal stem cells in the bone marrow, and this process acts on the adipose precursor cells such as insulin, insulin-like growth factor-1 (IGF-1) and growth hormone, ), Peroxisome proliferator activated receptor gamma (PPAR gamma), and the like. In particular, PPARγ, a nuclear receptor, plays a key role in adipocyte differentiation and does not produce adipose tissue during PPARγ deficiency. Ectopic overexpression of PPARγ induces adipocyte differentiation from fibroblasts.

In one embodiment of the present invention, the effect of inhibiting the differentiation of adipocytes was investigated by preparing a casein extract and a naked barley extract according to the heat treatment conditions and the type of the extracting solvent (Example 1) Whereas in the case of extracting the extract from the barnaclea extract using distilled water as a solvent, a significant inhibition of differentiation was confirmed (Example 2). In addition, the expression of PPARγ, aP2, C / EBPα, and CD36 mRNA was confirmed to be decreased by using the extract of Aobarifugi extract (AHB210), which has the most excellent effect of inhibiting adipocyte differentiation (Example 4). In another embodiment of the present invention, 8-week-old C57BL / 6 mice in which obesity was induced by high-fat diet were examined for body weight loss and low dietary efficiency according to ingestion of the bovine extract (see Example 5) (Example 6). The body weight of the obesity-induced rats was determined according to the ingestion of the bovine corn extract.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. In addition, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method.

Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose , Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dosage level is determined depending on the type of disease, severity, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition according to the present invention may vary depending on the age, sex, condition, body weight, absorbency, inactivation rate, excretion rate, type of disease, May be administered at a minimum of 2 weeks to at most 12 months apart, at least 1 to up to 20 times or more, and 1 × 10 ⁶ to 1 × 10 ⁸ cells may be administered at a dose of 1 × 10 ⁶ to 1 × 10 ⁸ cells And may be administered once or several times a day.

The pharmaceutical composition of the present invention may be administered to a subject in various routes. All modes of administration may be expected, for example, by subcutaneous, intravenous, intramuscular, or intraperitoneal injection. The pharmaceutical composition of the present invention is determined depending on the kind of the active ingredient, together with various related factors such as the disease to be treated, the route of administration, the age, sex, and weight and disease severity of the patient.

In another aspect of the present invention, the present invention provides a method for treating obesity comprising the step of administering the pharmaceutical composition to a subject. The term "individual" as used herein refers to a subject in need of treatment for a disease, and more specifically refers to a human or non-human primate, mouse, rat, dog, cat, It means mammals.

As another embodiment of the present invention, there is provided a food composition for preventing or ameliorating obesity, which comprises an extract of Bokbori water as an active ingredient.

As used herein, the term "improvement" means all actions that at least reduce the degree of symptom associated with the condition being treated. At this time, the functional food composition may be used either simultaneously with or separately from the agent for treatment before or after the onset of the disease to prevent or ameliorate obesity.

The term "health functional food composition" used in the present invention includes at least one of carriers, diluents, excipients and additives, and is formulated into one selected from the group consisting of tablets, pills, powders, granules, powders, . Examples of foods that can be added to the extract of the present invention include various foods, powders, granules, tablets, capsules, syrups, drinks, gums, tea, vitamin complexes, and health functional foods. Examples of the additive that can be further included in the present invention include natural carbohydrates, flavors, nutrients, vitamins, minerals (electrolytes), flavors (synthetic flavors, natural flavors and the like), colorants, fillers, , At least one component selected from the group consisting of an inorganic acid and a salt thereof, an organic acid, a protective colloid thickening agent, a pH adjusting agent, a stabilizer, a preservative, an antioxidant, glycerin, an alcohol, a carbonating agent and a pulp. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. As the above-mentioned flavors, natural flavors (tautatin, stevia extract (for example, rebaudioside A and glycyrrhizin) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used. The composition according to the present invention can be used in various forms such as flavorings such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies, factic acid and its salts, alginic acid and its salts, , A pH adjusting agent, a stabilizer, a preservative, a carbonating agent used in glycerin, an alcohol, a carbonated beverage, etc. In addition, the composition according to the present invention may contain flesh for the preparation of fruit juice and vegetable drinks . These ingredients may be used independently or in combination. Specific examples of the carrier, excipient, diluent and additives include, but are not limited to, lactose, dextrose, Cellulose, calcium carbonate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylquilolidone, cellulose, polyvinylpyrrolidone, methyl cellulose, water, sugar, starch, starch, It is preferable to use at least one selected from the group consisting of syrup, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

Example  1. Manufacture of Baejobori Extracts and Barley Extracts

Barley was largely classified into two types, ie, a barn and a barley. In order to compare the anti - obesity effects of different barley types and manufacturing processes, the barn and barley extracts were prepared. Covered rice was prepared by using the sample provided by Mr. Harak. First, in order to reduce the odor of barley, heat treatment was performed completely without heat treatment, or heat treatment was performed under the three conditions of 170 ° C, 210 ° C and 250 ° C for 20 minutes. 1.

More specifically, the covering and barbie were extracted with two solvents, ethanol and distilled water, depending on the type of solvent. Ethanol extraction was carried out in barley samples with 1: 2 (w / v) of 70% ethanol for 2 hours. Distilled water was extracted with barley at 90 ℃ distilled water at 1: 2 (w / v) for 30 minutes . The eight extracts and eight extracts were filtered with Whatman filter paper No. 2 and the filtrate was concentrated under reduced pressure to remove the solvent. Samples were recovered by distilled water and lyophilized.

(AHB, EHB, AHB170, EHB170, AHB210, EHB210, AHB250 and EHB250) and eight kinds of barberry extracts (ANB, ENB, ANB170, ENB170, ANB210, ENB250).

Example  2. Fat cells of the bark extract adipocyte ) Differentiation Inhibition  Confirm

2-1. Bokbori extract and barley Between extracts  Adipocyte differentiation Inhibition  compare

C3H10T1 / 2 cells were cultured with the above-mentioned Bacillus subtilis or Bacillus subtilis extracts to confirm adipocyte differentiation level in order to confirm the inhibitory effect of the 8 kinds of Bacilli and 8 kinds of Bacillus subtilis extracts of Example 1 on adipocyte differentiation.

More specifically, the C3H10T1 / 2 cell line derived from mouse embryonic fibroblasts was cultured in DMEM medium supplemented with 10% FBS, 1% penicillin and streptomycin at 37 ° C in a 5% CO 2 environment. C3H10T1 / 2 cells were cultured in a medium containing 1 uM dexamethasone, 5 ug / ml insulin, 20 nM 3-isobutyl-1-methylxanthine (IBMX) and PPARγ ligand (GW7845) for adipocyte differentiation at a concentration of 2.5 × 10 4 / / ml, 25 ug / ml, and 50 ug / ml, respectively, and incubated for 6 days. In order to visualize the degree of differentiation of adipocytes, the cells were stained with Oil Red O (Sigma-Aldrich, St Louis, MO, USA), which specifically reacted to lipids of differentiated cells. The cells were washed once with phosphate buffer saline (PBS) and fixed with 4% formaldehyde solution for 1 hour. The remaining fixative was removed and cells were stained with 0.2% Oil Red O solution for 1 hour. The excess of the staining reagent was removed and washing with distilled water was repeated to prevent the staining solution from remaining on the plate and dried.

As shown in FIG. 2 and FIG. 3, when compared with the control group CLA (conjugated linoleic acid), the inhibitory effect on the differentiation was not confirmed in the naked barley extract, while the effect of inhibiting the differentiation in the curd extract was confirmed. In addition, it was confirmed that, when extracted from 8 kinds of Baechuri extract as distilled water as a solvent, it inhibited the differentiation of C3H10T1 / 2, a stem cell. Among these extracts, AHB210, which was heat-treated at 210 ° C for 20 minutes, showed excellent inhibitory effect on adipocyte differentiation. The ethanol extract or AHB250 (AHB250), which was heat-treated at 250 ° C for 20 minutes, There was little or no.

2-2. Crest Water extract  Adipocyte differentiation Inhibition  Confirm

3T3-L1 cells were treated with a water extract (AHB210) of the bovine corn borneol treated at 210 ° C for 20 minutes, which confirmed the most excellent adipocyte differentiation inhibitory effect in Example 2-1, and the degree of adipocyte differentiation was confirmed.

(Dulbecco's Modified Eagle's Medium (DMEM) (Hyclone, Logan, UT, USA) and calf serum (CS) were used for maintenance of the lipogenic precursor cell 3T3-L1 (American Type Culture Collection, Rockville, (Hyclone, Logan, UT, USA) and 1% penicillin-streptomycin (Hyclone, Logan, UT, USA) were used in combination with 3T3-L1 differentiation and C3H10T1 / 2 USA) were mixed with Dulbecco's Modified Eagle's Medium (DMEM), 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) and 1% penicillin-streptomycin for cell culture and differentiation. (Sigma-Aldrich, St. Louis, Mo., USA), 5 μg / mL insulin (Sigma-Aldrich, St. Louis, Mo., USA) and 1 mM dexamethasone Sigma-Aldrich, St Louis, MO, USA). When the adipocytes became confluent at 37 ℃ and 5% CO ₂ incubator, adipocyte differentiation was induced and the culture medium was changed after 2 days. After that, the culture medium was changed every 3 days. DMS and IBMX were not added. It was differentiated for 4 ~ 7 days when the difference of the adipocyte differentiation was the maximum. In order to confirm the degree of differentiation of adipocytes, visualization was performed in the same manner as described in Example 2-1.

As shown in FIG. 4, it was confirmed that 3T3-L1, which is a lipid precursor cell, inhibited adipocyte differentiation when treated with a water extract (AHB210) of Bokbori treated at 210 ° C for 20 minutes.

(AHB210), which had been treated with high antioxidant activity and little odor at 210 ℃ for 20 min.

Example 3. Cytotoxicity analysis by MTT assay

(MTT) (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) (Sigma-Aldrich) was investigated to determine the cytotoxicity of AHB210 The number of viable cells was confirmed indirectly. When 3T3-L1 adipocytes were subconfluent in a 24-well plate, 5 mg / mL MTT reagent was added at 24 and 48 hours after extract treatment. After incubation for 4 hours, the medium was removed, dissolved in DMSO, and the absorbance was measured at 540 nm.

As shown in FIG. 5, no significant decrease in 3T3-L1 lipoprotein cell survival rate was observed in the AHB210 extract, which was heat-treated at 210 ° C for 20 minutes, and thus it was found to be harmless to the cells.

Example 4. Analysis of adipocyte differentiation factor expression through real-time RT-PCR

In order to confirm the reduction of expression of adipocyte differentiation factor by treatment with AHB210 treated with 210 ° C for 20 minutes, 3T3-L1 and C3H10T1 / 2 cells were supplemented with medium containing adipogenic differentiation medium and bovine extract The relative mRNA expression levels of PPARγ, aP2, C / EBPα and CD36 were examined by real-time RT-PCR after 6 days of treatment.

Total RNA was extracted and isolated from C3H10T1 / 2 cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Complementary DNA (cDNA) was synthesized using 0.5 μg RNA with the AMV Reverse Transcription System Kit (Promega, Madison, WI, USA) and random primers. PCR amplification (PCR) using 25 μL of the amplification mixture containing the primer and the Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) using Thermal Cycler Dice (Takara, Shiga, Japan) . Expression levels were normalized by 36B4 and all real-time PCRs were performed at least twice. The ΔCT value was calculated for the difference between the CT value of each sample and control (36B4) (ΔCT = CT (target) -CT (control)). Relative expression levels were calculated as 2- ^ΔCT .

As shown in FIG. 6, mRNA expression levels of PPARγ, aP2, C / EBPα, and CD36 were significantly decreased when treated with 12.5, 25, and 50 ug / ml of the extracts of heathen treated at 210 ° C. for 20 minutes Respectively.

Example 5. Confirmation of obesity inhibitory effect in obese mice induced by high fat diet

8 weeks old C57BL / 6 mice were purchased for 1 week to observe the effects of high - fat diets on obesity induced obesity. Control group (HFD) fed high fat diets and HFD + bovine group treated with high - fat diets were administered to the abdominal cavity at 210 ℃ for 20 min.

As shown in FIG. 7 to FIG. 12, a significant weight loss effect and a low food efficiency (FER) of the HFD + burden group were confirmed as compared with the HFD group. In addition, fat pad and liver showed a significant difference in long term weight. H & E (Hematoxylin and eosin) staining showed liver fat accumulation decreased by high fat diet. In the blood analysis results, the decrease of the factors such as cholesterol, glucose, triglyceride, and FFA related to obesity was also confirmed. In addition, high insulin sensitivity was confirmed by insulin sensitivity test.

Example 6. Confirmation of obesity inhibitory effect in obesity induced by hormone deficiency

Ovariectomy (OVX) was performed in rats to determine the effect of the bovine extract on obesity that could be induced in the menopause. At this time, the lets used in the experiment were 10-week-old SD rats (Oriental Bio), which were treated with side resection alone, ovariectomized sham group, ovariectomized OVX group, (AHB210), which was heat-treated at 210 ° C for 20 minutes, was divided into OVX + control group for 8 weeks.

As shown in Figure 13, significant weight loss of the OVX + control group was observed compared to the ovariectomized OVX group.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (6)

A pharmaceutical composition for preventing or treating obesity by inhibiting adipocyte differentiation and inhibiting fat accumulation, which comprises a water extract of a barnaclea bark heated at 210 占 폚 as a single active ingredient.
The method according to claim 1,
Wherein said obesity is due to hormone deficiency due to high fat diets or menopause.
delete The method according to claim 1,
Wherein said composition reduces mRNA expression of one or more genes selected from the group consisting of PPARy, aP2, C / EBPa, CD36.
A health functional food composition for preventing or ameliorating obesity by inhibiting adipocyte differentiation and inhibiting fat accumulation, which comprises a water extract of a barnaclea which is heat-treated at 210 占 폚 as a single active ingredient.
6. The method of claim 5,
Wherein said obesity is due to hormone deficiency due to high fat diets or menopause.

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