KR101503834B1 - A composition comprising a crude extract or purified fraction extract of Plantago asiatica for treating or preventing hyperlipidemia and obesity - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating hyperlipidemia and obesity containing an effective ingredient of a herbal extract or fraction. The herbal medicine extract of the present invention has an effect of inhibiting the lipid differentiation of 3T3-L1 cells, which are adipose cells derived from mouse, and the effect of decreasing body weight and fat, reducing blood lipid levels and histopathological effects in obesity-induced rats using high- Therefore, it can be usefully used as a medicament for preventing or treating hyperlipemia and obesity or a health functional food.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating hyperlipidemia and obesity, which contains, as an active ingredient, a herbal extract or a fraction thereof. [0002] The present invention relates to a composition for preventing or treating hyperlipidemia and obesity,

The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating hyperlipidemia and obesity which comprise an extract of fractions or fractions as an active ingredient.

[1] Kim DM, et al., Prevalence of obesity in Korea. Obes Rev. 6 , pp. 117-121, 2005

[Document 2] Kopelman PG. Obesity as medical problem. Nature 404: 635-643, 2000

[Literature 3] Kissebah AH, Vydelingum N, Murry R, et al. Relation of body fat distribution to metabolic complications of obesity. 54 (2): 254-60, 1982

[4] Van der Ploeg LH. Related Obesity: an epidemic in need of therapeutics. Curr Opin Chem Biol. 4 (4) , pp. 452-460, 200

[Reference 5] Zhang Xiangbian.張氏 類 經. Seoul, Korea. p.586, 1982

[Literature 6] Kim, EunSeon2, Obesity Therapeutics, In-depth Information Analysis Report, Korea Institute of Science and Technology Information, pp.1-111, 2002

[Reference 7] Mason EE. Methods for voluntary weight loss and control. Obes Surg 2: 275-276, 1992

[8] Park, B. G., Lee, H. S., et al.,: Single & 14-day repeated oral toxicity study and genotoxicological safety estimate of plantamajoside isolated from Plantago asiatica. J Toxicol Pub Health. 23 (1), 79-86, 2007

[Literature 9] Murase T. et al., Nootkatone, a characteristic constituent of grapefruit, stimulated energy metabolism and prevents diet-induced obesity by activating AMPK, Am J Physiol Endocrinol Metab, 299 (2) , pp.E266-75, 2010 )

[Literature 10] Wolfe K. et al., Antioxidant activity of apple peels. J. Agric. Food. Chem. 51 , pp. 609-614, 2003

[11] Lee JS et al., Supplementation of whole persimmon leaf improves lipid profiles and suppresses body weight gain in rat fed high fat diet, Food and Chemical Toxicology, 44 (11) , pp.1875-1883, 2006

Huang B. et al., Cinnamaldehyde inhibits adipocyte differentiation and adipogenesis via regulation of peroxisome proliferator-activated receptor-γ (PPARγ) and AMP-activated protein kinase (AMPK) pathways, J Agric Food Chem, Apr 27 (8) , pp.3666-73, 2011

[Literature 13] Allain C. et al., Enzymatic determination of total serum cholesterol, Clin. Chem. 20 (4) , pp.470-475, 1974

[14] Van Handel E. et al., Micromethod for the direct determination of serum triglycerides, J. Lab. Clin. Med. 50 (1) , pp.152-157, 1957

[Document 15] Warnick GR et al., Dextran sulfate-Mg 2 + precipitation procedure for quantitationof high density lipoprotein cholesterol, Clin. Chem. 20 (6) , pp.1397-1388, 1982

[16] Giusti G. et al., A comparative study of some spectrophotometric and colorimetric procedures for determination of serum glutamic-oxaloacetic and glutamic-pyruvic transaminase in hepatic diseases. Enzymol. Biol. Clin (Basel), 10 (1) , pp. 17-38, 1969

[17] DiGirolamo M. et al., Qualitative regional differences in adipose tissue growth and cellularity in male Wistar rats fed ad libitum. Am. J. Physiol. 274 , pp.R1460-1467, 1998

Today, modern people are getting richer and more delicious than others, but suffer from over calories due to high calorie, high fat intake and various chronic diseases. The most basic cause of adult illness is obesity. According to the Korea Institute of Health Policy in 2007, the number of diabetes deaths in Korea was 35.3, which is 2.5 times higher than the average of 13.5 in OECD member countries. The prevalence of type 2 diabetes mellitus is increasing rapidly in the 40s and 50s, and it is expected to continue to increase in the future (Kim DM, et al., Prevalence of obesity in Korea, Obes Rev. 6 , pp.117 -121, 2005).

Obesity is a disease caused by accumulation of excessive body fat due to unbalance of body energy consumption and consumption. It is not only a physical disease such as cardiovascular disease, diabetes, hypertension, hyperlipemia, gallstone disease, but also mental health. (Kopelman PG, Obesity as medical problem., Nature 404: 635-643, 2000). Especially in Asians, obesity management is more important because the body mass index is at least abdominal obesity more than Western people, and is highly susceptible to complications due to arterial diseases such as hypertension, diabetes and hyperlipidemia. The risk of developing obesity in obese people is 4 times higher in hypertensive patients and 10 times higher in diabetic patients than in normal people, and the mortality rate is expected to increase by 39% when the weight is over 25%. Therefore, the development of methods for prevention or treatment of obesity is urgently required. Obesity is a condition in which excessive fat accumulation occurs in the adipocyte due to the expression of a related gene, the action of metabolic hormones such as insulin, smoking, eating habits, activity and amount of exercise, and other control actions. (Kissebah AH, Vydelingum N, Murry R, et al., Relation of body fat distribution to metabolic complications of obesity 54 (2): 254-60, 1982).

In Oriental medicine, the cause of obesity is 粱粱 珍味, 气虛, 濕 滯, etc., and the treatment methods are water treatment, phlegmatism, heat conduction, active blood drug therapy is used (Zhang Xiang, Zhang, P. 586, 1982). Obesity treatment methods include the use of appetite suppressants, fat absorption inhibitors, energy metabolism accelerators, hormone preparations, and other surgical procedures such as gastrectomy and liposuction. However, (Van der Ploeg, LH, et al. ) , An epidemic in need of therapeutics, Curr Opin Chem Biol 4 (4) , pp. 452-460, 2004; Report, Korea Institute of Science and Technology Information, pp.1-111, 2002). Sibutramine, an appetite suppressant, and olestatet, a fat absorption inhibitor, are now recognized as long-term obesity drugs in the medical community, but there are still questions about side effects. In addition to drug administration and surgery to reduce body weight, diet, exercise, and behavior modification therapies are effective, and dietary therapy is the most important and fundamental method for the prevention and treatment of obesity (Mason EE. Methods for voluntary weight loss and control. Obes. Surg. 2: 275-276, 1992).

Chaegoncho (Plantago asiatica) is a perennial herb belonging to Plantaginaceae. It is distributed in three genera of 300 species in the world and is also called plantain. It is widely known that the field grasses growing on the streets and the streets in each area have been used widely as folk remedies because of their pharmacological actions such as germane, antibacterial and vigorous action and have various physiological activities such as immunity regulation, antioxidant activity and anti- have. (Park, BG, Lee, HS, et al., "Single & 14-day"). Toxicol Pub. Health 23 (1), 79-86, 2007).

As described above, the herbal medicine is safe and has been used continuously in oriental medicine and folk remedies.

Therefore, the inventors of the present invention confirmed that treatment with 3T3-L1 adipocyte extract and fractions thereof inhibited cell differentiation. In addition, in the obesity-induced animal using the high-fat diet, not only the decrease in body weight and body fat, Leptin, and adiponectin in the blood. In addition, by observing the histopathological effects of liver, epididymis, fat and pancreas, the present invention can be utilized as an effective therapeutic agent for obesity.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating hyperlipemia and obesity, which comprises an extract of fragments or fractions as an active ingredient.

In addition, the present invention provides a health functional food for preventing or ameliorating hyperlipidemia or obesity, which comprises a tea extract or fraction as an active ingredient.

The crude extract as defined herein is an extract, preferably a water and ethanol mixed solvent, more preferably 30 to 100% (v / v) of an extract soluble in a solvent selected from water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, ) And extracts soluble in water and ethanol mixed solvent.

A fraction as defined herein is a fraction which is soluble in a nonpolar solvent selected from n-hexane, heptane, methylene chloride, chloroform or ethyl acetate, preferably a hexane or ethyl acetate solvent, more preferably ethyl acetate, and the nonpolar solvent soluble fraction Removed polar solvent, preferably a fraction soluble in butanol solvent.

The crude extracts and fractions of the present invention can be obtained as follows.

Hereinafter, the present invention will be described in detail.

After drying the bean gum of the present invention and crushing it, the mixture is pulverized to have a polarity of about 1 to 100 times, preferably about 2 to 20 times the weight of the sample, water, alcohol, lower alcohol having 1 to 4 carbon atoms, Preferably 20 to 120 ° C, preferably room temperature to 80 ° C for about 1 to 72 hours in a solvent, preferably a mixed solvent of water and ethanol, more preferably 30 to 100% (v / v) water and ethanol Preferably 2 to 12 hours, using an extraction method such as cold extraction, hot water extraction, cold extraction, reflux cooling extraction or ultrasonic extraction, preferably by a cold extraction method, followed by filtration under reduced pressure and concentration to obtain the herbal extract Are obtainable; A third step of adding the crude extract obtained in the above step to water in an amount of about 0.05 to 50 times, preferably 0.5 to 5 times (w / v) of the weight of the crude extract to prepare a suspension; The suspension is admixed with a non-polar solvent selected from n-hexane, heptane, methylene chloride, chloroform or ethyl acetate, about 0.5 to 20 times volume, preferably 1 to 5 times volume (w / v) Can be obtained by sequentially repeating the fractionation step by adding hexane or ethyl acetate solvent, more preferably ethyl acetate, to obtain the non-polar solvent-soluble fraction and the polar solvent-soluble fraction of the present invention, respectively.

The present invention provides a pharmaceutical composition and a health functional food for the treatment or prevention of hyperlipidemia and obesity comprising the above-mentioned production method and a tea extract or fraction obtained by the above-mentioned manufacturing process as an active ingredient.

The herbal extracts and fractions obtained from the above showed a reduction in lipid differentiation of adipocytes and showed a reduction in body weight, body fat and blood lipid levels in obesity-induced rats using a high fat diet.

The pharmaceutical composition for the treatment of hyperlipidemia and obesity of the present invention comprises 0.01 to 80% by weight, preferably 10 to 50% by weight of the above extract, based on the total weight of the composition.

The pharmaceutical compositions comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The pharmaceutical dosage forms of the extract of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in suitable aggregates.

The pharmaceutical composition containing the extract according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols or the like, oral preparations such as external preparations, suppositories, Can be used. Examples of carriers, excipients and diluents that can be included in the composition containing the extract 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. In the case of formulation, a diluent or excipient such as a filler, a weighting agent, a binder, a wetting agent, a releasing agent, a surfactant and the like is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, (sucrose), lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol gelatin and the like can be used.

The preferred dosage of the pharmaceutical composition comprising the extract of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the pharmaceutical composition containing the extract of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The herbal ethanol extract of the present invention can be administered to mammals such as rats, mice, livestock, and humans in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injection.

The present invention also provides a health functional food for preventing or ameliorating hyperlipidemia and obesity, which comprises, as an active ingredient, a tea extract or fraction having an excellent inhibitory effect on obesity.

&Quot; Health functional food "as defined herein means food prepared and processed using raw materials or ingredients having functionality useful to the human body in accordance with Law No. 6727 on Health Functional Foods." Functional " Structure and function of the nutrient to control or physiological effects, such as to obtain a beneficial effect for health is intended to eat.

The health functional food for preventing or ameliorating hyperlipemia and obesity according to the present invention contains 0.01 to 95% by weight, preferably 1 to 80% by weight, of the above extract relative to the total weight of the composition.

For the purpose of preventing or ameliorating hyperlipidemia and obesity, a pharmaceutical dosage form such as a powder, granule, tablet, capsule, pill, suspension, emulsion or syrup or a health functional food in the form of a tea bag, Manufacturing and processing are possible.

In addition, the present invention provides a health supplement containing as a main ingredient a herbal extract or fraction showing a hyperlipidemia and an obesity-suppressing activity.

In addition, the present invention provides a food additive containing as a main ingredient a herbal extract or fraction showing hyperlipidemia and obesity-suppressing activity.

The health functional beverage composition of the present invention has no particular limitation on the other ingredients other than the above-mentioned extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. 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 natural flavors other than those described above, natural flavors (such as tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin)) and synthetic flavors (saccharin, aspartame, etc.) have. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the extract of the present invention can be added to foods or drinks for the purpose of preventing hyperlipidemia and obesity. At this time, the amount of the extract in the food or drink may be 0.01 to 15% by weight of the total food, and the health beverage composition may be added in a proportion of 0.02 to 5 g, preferably 0.3 to 1 g, .

The herbal ethanol extract of the present invention inhibits lipid differentiation in 3T3-L1 adipocyte derived from mouse embryo fibroblast and is effective in reducing weight loss and body fat, blood lipid levels in obesity-induced rats Leptin and adiponectin as well as to reduce the blood concentration of leptin and adiponectin and is also effective in the histopathological effect, so that it can be usefully used in pharmaceutical compositions, health functional foods and health supplement foods for prevention or treatment of obesity have.

1 is a graph showing the effect of CCE and CCE fractions on MTT assay of 3T3-L1 cell line of adipocytes,
FIG. 2 is a graph showing the effect of CCE on the tunneling of 3T3-L1, a cell line of adipocytes,
FIG. 3 (ab) is a graph showing the effect of CCE and CCE fractions on Oil Red-O staining of 3T3-L1, a cell line of adipocytes,
FIG. 4 is a graph showing the effect of CCE on the body weight and dietary level of obese rats due to administration of high fat diet,
FIG. 5 is a graph showing the effect of CCE on the liver weight of obese rats due to administration of a high fat diet,
6 (a) shows the effect of CCE on the body fat weight of obese rats due to administration of high fat diet,
FIG. 7 (ad) is a graph showing the effect of CCE on blood lipid concentration in obese rats due to administration of high fat diet,
FIG. 8 is a graph showing the effect of CCE on liver tissue diameter in obese rats due to administration of a high fat diet system,
FIG. 9 is a graph showing the effect of CCE on the epididymal tissue diameter of obese rats due to administration of high fat diet,
FIG. 10 is a graph showing the effect of CCE on the pancreatic tissue diameter of obese rats due to administration of the high fat diet system,
11 is a graph showing the effect of CCE on insulin-producing cells in the pancreatic tissue diameter of obese rats due to administration of a high fat diet system,
FIG. 12 is a graph showing the effect of CCE on glucagon-producing cells in the pancreatic tissue diameter of obese rats due to administration of high fat diet,
13 is a graph showing the effect of CCE on expression of AMPK and ACC protein in muscle tissue of obese rats due to administration of high fat diet.

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following Examples, Reference Examples and Experimental Examples.

Example 1. Preparation of herbal extracts

The tea used in the experiment was purchased from the Ministry of Health and Welfare (Seoul, Korea) and differentiated and selected from Daegu Hanyang University 's herbal medicine classroom. It was cleaned with distilled water and then used for experiments. Ten grams of 95% ethanol was added to 600 g of dry herbal medicine, incubated at room temperature for 2 hours, and then filtered with 2-layer gauze and Wattman 3M paper (1440-320). The collected filtrate was concentrated under reduced pressure using a rotary evaporator (N-1110V, EYELA, Japan) and freeze-dried to obtain 51.6 g (8.6%) of the dried product (hereinafter referred to as CCE).

Example  2. Chinchon  Ethanol extract Fraction  Produce

The tea used in the experiment was purchased from the Ministry of Health and Welfare (Seoul, Korea) and differentiated and selected from Daegu Hanyang University 's herbal medicine classroom. It was cleaned with distilled water and then used for experiments. The extract was extracted with 95% ethanol (6L × 3 times) and then concentrated under reduced pressure on a rotary evaporator (N-1110V, EYELA, Japan) to obtain 53.0 g of ethanol concentrate. This was dispersed in 1000 ml of distilled water and extracted with methylene chloride (CH ₂ Cl _2, 1 L X 3 times) and concentrated to obtain 9.5 g of a methylene chloride fraction (hereinafter referred to as CH ₂ Cl ₂ ). The remaining aqueous layer was extracted with ethyl acetate (EtOAc, 1 LX 3 times) and the extracts were concentrated to give 3.3 g of ethyl acetate fraction (hereinafter referred to as EtOAc). Subsequently, the aqueous layer was extracted again with butanol (n-BuOH, 1 L X 3 times) and concentrated to obtain 8.3 g of a butanol fraction (hereinafter referred to as n-BuOH). The aqueous layer was further concentrated to obtain 30.4 g of a water fraction Quot;).

Reference Example 1. Preparation of experimental material

3T3-L1 mouse embryonic fibroblasts were purchased from the American Type Culture Collection (ATCC, CL-173TM), Dulbeccos modified Eagles media (DMEM, SH30243.01), Bovine calf serum (BCS, SH30401.01) , Fetal bovine serum (FBS, SH30396.03) and penicillin / streptomycin (PS) were purchased from Hyclon (SV30010, Hyclone, USA). (DMSO, D2650), Oil Red-O (O0625-25), dexamethasone (DEX, D4902-100MG) and isobutyl methyl Isobutylmethylxanthine (IBMX, I5879-250MG) was purchased from Sigma-Aldrich company (St. Louis, MO, USA).

Reference Example 2. Preparation of experimental animals

Five-week-old male Sprague Dawley rats weighing 180 g were randomly assigned to one cage at a temperature of 22 ± 2 ° C, humidity of 53.3%, light-dark at 12:12 (Hyochang Science Co., Ltd.) and water. All the experimental animals were purified for 1 week after purchase and then divided into 3 groups.

Reference example   3. 3 T3 - L1  Cell culture

3T3-L1 mouse mouse embryo fibroblast was cultured by the following method (Lin J. et al., Green Tea Polyphenol Epigallocatechin Gallate Inhibits Adipogenesis and Induces Apoptosis in 3T3-L1 Adipocytes, Obesity Research, 13 (6) , pp.982-990, 2005).

First, the cells were cultured in Dulbecco's modified Eagle's medium (SH30243.01) containing 10% bovine calf serum at 37 ° C in a 5% CO ₂ incubator (Day 0). After 8 days of culture, preadipocytes were transferred to DMEM medium (167 nM insulin, 0.5 μM isobutylmethylxanthine, 1 μM dexamethasone) containing 10% fetal bovine serum (FBS) for 2 days (Day 2) . 167 nM insulin for 2 days and then cultured in 10% FBS / DMEM for 4 days to produce mature adipocytes.

Reference example  4. Chinchon  Administration of ethanol extract and induction of obesity

In order to induce obesity, experiments were carried out using the dietary composition described in the literature (Lee JS et al., Supplementation of whole persimmon leaf improves lipid profiles and suppresses body weight gain in rat high-fat diet, Food and Chemical Toxicology, 44 (11) , pp. 1875-1883, 2006).

High-fat diets containing 15% (lard-10% w / w, con oli-5% w / w) of carbohydrates according to the diet of Korean stocks (# 102038, AIN-76A Based High Fat / High Carb Purified Rodent Diet (Cholesterol and Cholic Acid, Dyet, USA). The fat content of fat was reduced by 30% and the calories of carbohydrate were increased by 50%. The experimental group was divided into 4 groups of 15 animals per group. The normal diet (AIN-76) and the high fat diet (HF) fed high fat diets and saline to NOR group. In the experimental group, high - fat diet and herbal extracts (CCE) were orally administered at 300 mg / kg for 8 weeks.

Ingredient Normal HF Casein 200 200 DL - Methionine 3 3 Com starch 150 150 Sucrose 500 397.5 Cellulose 50 50 Corn oil 50 50 Mineral-mix 35 35 Vitamin-mix 10 10 Choline bitartrate 2 2 Lard - 100 Cholesterol - 2 Cholic acid - 0.5 total (g) 1000 1000 Kcal / 100g diet 3850 4340 Calorie from fat (%) 11.7 31.1 Calorie from carbohydrate (%) 67.5 50.5 Calorie from protein (%) 20.8 18.4

Reference Example 5. Statistics Processing

The statistical significance of the test results was analyzed using two-way ANOVA and LSD post-test of the SPSS program (Version 14.01) . 05 , and the statistical significance (#) to the normal group and the statistical significance (*) to the treatment group (HF) were shown in the figures and tables, respectively.

Experimental Example 1. Cytotoxicity (MTT assay)

To investigate the effect of CCE on the 3T3-L1 cells, MTT assay was used (Lin J. et al., Green Tea Polyphenol Epigallocatechin Gallate Inhibits Adipogenesis and Induces Apoptosis in 3T3 -L1 Adipocytes, Obesity Research 13 (6), pp.982-990, 2005).

The MTT assay is a method for measuring the absorbance of blue formazan crystals produced by dehydrogenase in mitochondria of living cells by dissolving them in DMSO (dimethyl sulfoxide). The 3T3-L1 cell line cultured in Reference Example 1 was used, and cells in exponential growth phase were used in the experiment. In other words, 5 × 10 ⁴ cells / ml were mixed with 5% tryphan in the same volume for the same period without any treatment, and the number of living cells was measured. The viability was evaluated using the average value of three wells. After adding 50 μl of MTT solution (2 mg / ml) per well, incubate at 37 ° C in a 5% CO ₂ incubator for 2 hours, remove the medium and add 100 μl of DMSO per well. After dissolution, the absorbance was measured at 540 nm in an ELISA reader (Tekan, Austria, Switzerland).

As a result, as shown in FIG. 1-A, the survival rate of adipocytes was decreased in dependence on the concentration of CCE sample compared to the control treated with DMSO alone. Seemed not to give. In FIG. 1-B, the fat cell survival rate was also decreased in a concentration-dependent manner in the fraction of the CCE extract, but it was confirmed that the fraction had no significant effect on the sample treatment concentration.

Experimental Example 2: Tunnel staining using cells

TUNEL staining was performed using the TUNEL assay kit (11684795910, Roche, Manheim, Germany) to observe apoptosis-positive cells.

Cells were cultured in 4 chamber slides and differentiated. 50 ug / ml of sample and 200 ug / ml of CCE were treated for 24 hours at the end of differentiation and treated with 0.5 mM Rosiglitazone (R2408, Sigma-Aldrich, USA) Quot;). The cells were washed with D-PBS (SH30028.03, Thermo, USA), fixed with 4% paraformaldehyde for 1 hour at room temperature, washed again with D-PBS and blocked with 3% H ₂ O ₂ ), Permeabilization was performed by adding permeabilization solution, and 50 μl of TUNEL assay kit (11684795910, Roche, Manheim, Germany) was added and reacted in a dark room at 37 ° C for 1 hour and stained with DAB solution.

As a result, as shown in FIG. 2, apoptotic cells were not observed in the control group treated with DMSO alone, but adipocyte apoptosis was induced in the 0.5 mM Rosiglitazone-treated group and the CCE group as positive control drugs.

Experimental Example 2: oil red-O  dyeing

To investigate the effect of supernatant ethanol extract (CCE) on adipocyte differentiation and lipogenesis in 3T3-L1 cells, an oil red-O staining experiment was performed (Lin J. et al., Green Tea Polyphenol Epigallocatechin Gallate Inhibits Adipogenesis and Induces Apoptosis in 3T3-L1 Adipocytes, Obesity Research 13 (6), pp.982-990, 2005).

Oil Red-O staining is a method of measuring the fat produced in the cells by staining with differentiated 3T3-L1 cells with Oil Red-O reagent. The cells were washed with D-PBS (DPBS, SH30028.03), fixed with pH 7.2 Cacodylate buffer for 2 hours, and stained with Oil-red O Solution. After cell staining, the cells were washed three times with 40% isopropyl alcohol (2-propanol, 67-63-0) and dried. The intracellular fat was measured with an optical microscope (AE 31, Motic, USA).

As shown in FIG. 3-A, during the differentiation of 3T3-L1 cells, CCE extracts were treated at a concentration of 100 μg / ml (B), 150 μg / ml (C) and 200 μg / As a result of the treatment of the samples, the inhibition of cell differentiation and lipogenesis was inhibited in a concentration dependent manner compared with the control (control). As a result of observation of each herbal extract of each dose with a microscope and naked eye, CCE extract showed dose- And it inhibited the differentiation and fat accumulation of adipocytes. As shown in FIG. 3-B, the fractions of CCE extract, methylene chloride (CH ₂ Cl ₂ ), ethylacetate (EtOAc), butanol (BuOH) and water (H ₂ O) fractions were each dissolved at a concentration of 200 μg / (EtOAc) and butanol (BuOH) fractions were measured in the intracellular fat (P <0.05). The results showed that the degree of staining of intracellular fat was 41.30%, 29.46%, 108.58% and 133.41% The inhibition of accumulation, methylene chloride (CH ₂ Cl ₂ ), and water (H ₂ O) fractions were found to increase fat accumulation.

Experimental Example 3 Dietary Efficacy, Body Weight and Tissue Harvesting in Obese-Induced Rats Using High Fat Diet

Dietary intake was measured daily in the morning, daily intake was calculated, and body weight was measured once a week. The food efficiency ratio (FER) is the body weight gain during the experiment and the value divided by the dietary intake (FER = body weight gain / food intake).

As shown in FIG. 4 and Table 2, the HF group showed a significant increase in body weight compared with the NOR group and the CCE group after 2 weeks from the start of the diet. On the other hand, the NOR group and the CCE group showed significant weight loss (p <0.05) as compared with the HF group, and showed the same tendency until the end of the experiment by 8 weeks. In the CCE group, (p < 0.05). Therefore, CCE administration significantly inhibited the body weight gain in obese rats induced by high fat diet, which was more decreased than in the NOR group. Dietary intake was lower in the NOR group than in the HF group, but higher in the CCE group and higher in the HF group in terms of dietary efficiency. The changes in body weight gain were significantly ( p <0.05) increased in the HF group compared to the NOR group, and decreased in the CCE group compared to the HF group.

On the other hand, animals were fasted for 12 hours, anesthetized by inhalation of ether, blood was collected from the abdominal subclavian vein using EDTA-treated tubes, and centrifuged at 3000 rpm (4 ° C) for 15 minutes to separate the plasma Respectively. Blood was collected from the experimental animals, and hepatic and adipose tissues (epididymal fat, peritoneal fat, abdominal subcutaneous fat) were immediately removed and washed several times with 1X PBS buffer to remove moisture from the surface and weighed. Some liver and brain tissue were fixed in 10% formaldehyde solution and refrigerated. The remaining organs and fat tissue for protein expression analysis were immediately quenched into liquid nitrogen and stored at -70 ° C until analysis. Tissues were rapidly frozen in liquid nitrogen and stored at -80 ° C until used.

According to the results of FIG. 5 and FIG. 6 (AB), liver weight, epididymal fat mass, peripheral fat weight, internal weight and brown fat (BAT) were significantly increased in the HF group compared to NOR, Compared to the control group. This result shows that the effect of strong CCE on body fat reduction, which is known to cause complications such as cardiovascular disease rather than simple weight loss in obesity, is more meaningful as a therapeutic agent for obesity.

NOR HF CCE Food intake (g / day) 21.29 + 0.44 ^* 23.59 + - 0.44 ^# 24.02 + - 0.28 ^# Weight gain (g / day) 4.80 ± 0.06 ^* 6.14 ± 0.13 ^# 5.40 ± 0.19 FER 0.23 ± 0.00 0.26 ± 0.00 0.23 ± 0.00

Experimental Example  4. Obesity-induced In rats Chinchon  Effect of Ethanol Extract on Changes in Serum Lipid Concentration

Each group of animals was fasted for 12 hours before sacrifice and lightly anesthetized with CO ₂ , and blood was collected from the abdominal vena cava. The total blood cholesterol (TC) was measured by the enzyme method of C. Allain 1 (Allain 1), and the total cholesterol (TC) was measured by centrifugation at 4 ° C and 3,500 rpm for 15 minutes. (L-type GHO M, Wako, Japan) was used in accordance with the method described in K. et al., Enzymatic determination of total serum cholesterol, Clin. Chem. 20 (4) , pp.470-475, triglyceride content was determined by the enzyme method of Van Handel et al (Micromethod for the direct determination of serum triglycerides, J. Lab. Clin. Med. 50 (1) , pp. 152-157, 1957) (L-type TG M, Wako, Japan) was used. High density lipoprotein (HDL-cholesterol) and low density lipoprotein (LDL-cholesterol) levels of enzymatic Warnick (Warnick GR et al., Dextran sulfate-Mg 2+ precipitation procedure for quantitationof high density lipoprotein cholesterol, Clin. Chem. 20 (6) (Choesterol N HDL, Choesterol N LDL, SEKISUI, Japan) according to the manufacturer's instructions, pp.1397-1388, 1982). Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) contents, which are transaminases related to liver function, were determined by spectrophotometry (Giusti G. et al., A comparative study of some spectrophotometric and (L-type ALT) using an enzyme-linked immunosorbent assay (ELISA ) for the determination of serum glutamic-oxaloacetic and glutamic-pyruvic transaminase in hepatic diseases. Enzymol. Biol. (Hitachi 7080, Japan) using an automated biochemical analyzer (J2, L-type AST J2, Wako, Japan). The concentration of leptin in the serum was determined using a Leptin ELISA kit (900-015, Assay Designs, USA) and the concentration of adiponectin in the serum was measured using an Adiponectin ELISA kit (44-ADPR-0434, ALPCO Diagnostics, USA) ELISA reader (Tecan, Germany).

As shown in FIG. 7-AC, when plasma ALT levels were compared with NOR, there was a significant increase in the CCE group compared to the HF group. In the AST level, the HF group was higher than the NOR group, But there was no significant change. In the LDL-C level, NOR was significantly lower than HF, but also in the CCE group, and the CCE was significantly higher in HDL-C than in the HF group. Total cholesterol was significantly lower in the NOR and CCE groups than in the HF group. Glucose and TG levels were significantly decreased in NOR group and CCE group compared to HF group. Therefore, the 8-week high-fat diet showed an increase in LDL-C in proportion to the increase in lipid (total cholesterol, triglyceride) increased by ingestion, thereby increasing the induction rate of arteriosclerotic coronary disease, cerebrovascular disease or arteriosclerosis and complications . This indicates that CCE administration increases serum HDL-C levels and inhibits the production of LDL-C, thereby decreasing accumulated cholesterol in the blood vessels and improving the body's lipid metabolism. Therefore, high-fat diets increase the activity of ALT and AST in the liver due to metabolic stress, but this is reduced by administration of CCE, indicating that CCE administration can improve liver function.

As shown in FIG. 7-D, the serum level of adiponectin secreted from adipocytes while acting as an insulin-sensitizing hormone was lower than that of the high fat diet group (HF) in the normal group (NOR) The adiponectin content tended to be lower than the high fat diet group (HF). On the other hand, the serum level of leptin, which is known to be associated with appetite regulation as a peptide secreted from adipocytes, is higher in the high fat diet group (HF) than in the normal group (NOR) The leptin content of serum was significantly decreased by CCE administration than that of high fat diet group (HF).

Experimental Example  5. CCE  Administration HF  supply In the rat  Histopathological effects: evaluation of liver, epididymal fat and pancreas

Liver, epididymal fat, and part of pancreatic parenchyma were fixed and fixed in 10% neutral formalin, dehydrated and paraffin-embedded in the usual manner, and 3-4 μm sections were prepared and stained with hematoxylin-eosin. And insulin and glucagon-producing cells in the pancreatic islets were stained with avidin-biotin-peroxidase (ABC) method. (% / Mm2 of hepatic parenchyma), hepatocyte diameter (μm / hepatocyte), epididymal adipocyte cell size (μm / adipocyte) and pancreatic zymogen granules (% / mm2 of exocrine pancreatic parenchyma), number of islets / mm ² of pancreas, number of insulin and glucagon producing cells (cells / 200 μm ² of endocrine pancreas) and their ratio (insulin / glucagon cell ratio) And histomorphometry was performed using a DMI-300 Image Processing (DMI, Korea) in each of the microscopic fields.

As a result, as shown in FIG. 8, severe fatty liver was detected due to fatty change, which was characterized by hypertrophy and vacuolization of hepatocytes throughout the liver lobe by high fat feed, . In Fig. 9, it was recognized that hypertrophic adipocyte hypercellular hypertrophy was observed, but the diameter of adipocyte adipose pericytes was decreased by CCE group.

As shown in FIG. 10, a decrease in the pancreatic zymogen granule was observed due to the feeding of the high fat diet, but it was confirmed that the inhibition of the reduction of the zymogen granule of the pancreas was recognized in the CCE group. In Figs. 11 and 12, endocrine disorders such as increase of insulin and glucagon producing cells due to feeding of high-fat diet were recognized, but insulin / glucagon ratio was observed similar to NOR group in CCE group, Lt; / RTI &gt; As a result of this experiment, it was observed that the CCE group significantly inhibited the increase of the hepatic hepatopathy caused by HF supply, epididymal adipocyte hypertrophy, insulin and glucagon producing cells, and particularly suppressed decrease of zymogen granule of pancreas. And the control of lipid absorption and metabolism through the regulation of secretion of HF was suppressed, but the precise mechanism was not known.

Experimental Example  6. In the muscle AMPK , ACC Active

Western blot analysis was performed to determine the effect of CCE on the expression of AMPK, ACC, which is a protein related to energy and lipid metabolism, in obesity - related markers in obese - induced rats. (Murase T. et al., Nootkatone, a functional constituent of grapefruit, stimulated energy metabolism and prevents diet-induced obesity by activating AMPK, Am J Physiol Endocrinol Metab, 299 (2), pp. E266-75, 2010).

The muscle tissue isolated from the rats was suspended in lysis buffer (137 mM NaCl, 1% Triton X-100, 20 mM Tris-HCl (pH 7.4), 2 mM EDTA, 2 mM sodium pyrophosphate, 1 mM Na3VO4, 10% glycerol, / Ml leupeptin, 5 μg / ml aprotinin, 35 μM benzamidine, 0.5 mM DTT, 1 mM PMSF) and centrifuged at 14,000 g for 10 min at 4 ° C to collect the supernatant. The concentration of the separated proteins was quantitated using a BSA reagent (# 23209, PIERCE, USA), and 50 단백 protein was electrophoresed on 8% SDS-PAGE. Gel separated proteins were transferred to polyvinylidene difluoride membranes (PVDF, HATF09025, 0.45 mm, Millipore) and incubated with 5% bovine serum albumin (BSA) -TBST (TBS, 0.1% Tween-20) (# 3662, Cell Signaling, MA, USA), P-ACC (# 3661, Cell Signaling, MA, USA) and anti-AMPK The secondary antibody (sc-4777, goat) was incubated with the primary antibodies against the membranes after overnight incubation, respectively, using β-actin (905-733-100, anti-rabbit-HRP, 1: 5000 dilution, Santa Cruz Biotechnology, USA) for 1 hour at room temperature. After incubation with ECL reagent (# 34079, Amersham Biosciences, IL, USA) for 1 min, the target protein attached to the membrane was identified using Image Quant software (EW-97701-62, UVP, USA).

As shown in FIG. 13, the AMPK phosphorylation level was measured in order to measure the activity of AMPK enzyme, which is known to regulate cell metabolism and energy balance. As a result, phosphorylation level of AMPK protein was decreased in HF group compared to NOR group , And increased in the CCE group compared to the HF group. In addition, phosphorylation of ACC protein, which plays an important role in fatty acid metabolism, decreased ACC phosphorylation in HF group and increased in CCE group compared to HF group compared to NOR group.

Hereinafter, formulation examples of the composition containing the extract of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically explained.

Formulation example  One. Sanje  Produce

Herbal tea ethanol extract (CCE) ------------------------------------ 20 mg

Lactose ------------------------------------------------- ------- 100 mg

Talc ------------------------------------------------- -------- 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation example  2. Preparation of tablets

Herbal tea ethanol extract (CCE) ------------------------------------ 10 mg

Corn starch ------------------------------------------------ - 100 mg

Lactose ------------------------------------------------- ------- 100 mg

Magnesium stearate ------------------------------------------- 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example  3. Preparation of capsules

Herbal tea ethanol extract (CCE) ------------------------------------ 10 mg

Crystalline cellulose --------------------------------------------- 3 mg

Lactose ------------------------------------------------- - 14.8 mg

Magnesium stearate 0.2 mg &lt; RTI ID = 0.0 &gt;

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation example  4. Preparation of injections

Herbal tea ethanol extract (CCE) ------------------------------------ 10 mg

Mannitol ------------------------------------------------- ----- 180 mg

Sterile sterile distilled water for injection ----------------------------------------- 2974 mg

Na ₂ HPO ₄ , 12H ₂ O -------------------------------------------- ----- 26 mg

(2 ml) per ampoule in accordance with the usual injection method.

Formulation example  5. Liquid  Produce

(EtOAc) 20 mg &lt; RTI ID = 0.0 &gt;

Isseong Party ------------------------------------------------ ------ 10 g

Mannitol ------------------------------------------------- -------- 5 g

Purified water ------------------------------------------------- -------

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, And sterilized to prepare a liquid preparation.

Formulation example  6. Manufacture of health food

Herbal tea ethanol extract (CCE) ------------------------------------ 1000 mg

Vitamin mixture ------------------------------------------------ --- Suitable amount

Vitamin A Acetate ------------------------------------------- 70 [mu] g

Vitamin E ------------------------------------------------ ----- 1.0 mg

Vitamin B1 ------------------------------------------------ --- 0.13 mg

Vitamin B2 ------------------------------------------------ --- 0.15 mg

Vitamin B6 ------------------------------------------------ ---- 0.5 mg

Vitamin B12 ------------------------------------------------ --- 0.2 μg

Vitamin C ------------------------------------------------ ------ 10 mg

Biotin ------------------------------------------------- ------- 10 μg

Nicotinic acid amide 1.7 &lt; RTI ID = 0.0 &gt; Mg

Folic acid ------------------------------------------------- --------- 50 μg

Calcium Pantothenate ------------------------------------------------ 0.5 mg

Inorganic mixture ------------------------------------------------ - Suitable amount

Ferrous sulfate ---------------------------------------------- ----- 1.75 mg

Zinc oxide ------------------------------------------------ ---- 0.82 mg

Magnesium carbonate ------------------------------------------------ 25.3 mg

Potassium monophosphate ---------------------------------------------- ----- 15 mg

Calcium Phosphate ---------------------------------------------- ----- 55 mg

Potassium citrate ------------------------------------------------ ---- 90 mg

Calcium Carbonate ------------------------------------------------ ----- 100 mg

Magnesium Chloride ------------------------------------------------ 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation example  7. Manufacture of health drinks

The first fraction of ethacetate (EtOAc)

Citric acid ------------------------------------------------- ----- 1000 mg

oligosaccharide ------------------------------------------------- ----- 100 g

Plum concentrate ------------------------------------------------ ------ 2 g

Taurine ------------------------------------------------- --------- 1 g

Purified water was added to 900 ml

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

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Claims (6)

The bean curd is dried and ground, and then extracted with a cold extraction method at 20 to 120 ° C for 2 to 12 hours in a volume (w / v) water and ethanol mixed solvent of 2 to 20 times the weight of the sample, A first step of obtaining anchovy crude extracts; A second step of adding the crude extract obtained in step 1 to water in an amount of 0.5 to 5 times (w / v) of the weight of the crude extract to prepare a suspension; A pharmaceutical composition for the prevention or treatment of obesity comprising the fraction of ethacetate obtained by adding 1 to 5 times volume (v / v) ethyl acetate of the suspension to the suspension, as an active ingredient. delete delete The bean curd is dried and ground, and then extracted with a cold extraction method at 20 to 120 ° C for 2 to 12 hours in a volume (w / v) water and ethanol mixed solvent of 2 to 20 times the weight of the sample, A first step of obtaining anchovy crude extracts; A second step of adding the crude extract obtained in step 1 to water in an amount of 0.5 to 5 times (w / v) of the weight of the crude extract to prepare a suspension; A health functional food for preventing or ameliorating obesity comprising an effective amount of a fractionated fraction of ethacetate obtained by adding 1 to 5 times volume (v / v) ethyl acetate of the suspension to the suspension. delete delete

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