KR20170005632A

KR20170005632A - A composition comprising extract of Elaeagnus multiflora Thunb. for preventing or treating obesity or hyperlipidemia

Info

Publication number: KR20170005632A
Application number: KR1020150095940A
Authority: KR
Inventors: 김은정; 정광조; 김승형
Original assignee: 김은정
Priority date: 2015-07-06
Filing date: 2015-07-06
Publication date: 2017-01-16

Abstract

The present invention relates to a composition comprising an Elaeagnus multiflora Thunb. extract for preventing or treating obesity or hyperlipidemia. The Elaeagnus multiflora Thunb. extract has an excellent effect of degrading adipose differentiation and accumulation of fat in adipocytes, and activity of reducing the level of total cholesterol or low density lipoprotein cholesterol in a serum, thereby being useful for a composition for preventing or treating obesity or hyperlipidemia.

Description

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

The present invention relates to a composition for preventing or treating obesity or hyperlipidemia comprising extract of Elaeagnus multiflora Thunb.

Obesity is not only a problem of lowering the quality of life and the cosmetic effects of overweight, but also of the problems of hyperlipidemia, type 2 diabetes, coronary artery disease, degenerative arthritis, prostate, breast cancer, colon cancer, endometritis, sleep apnea and respiratory disorders, It is closely related to major causes of death such as gallstone disease, depression, and dyslipidemia (Na et al., 2009).

According to a report released by the International Obesity Council, one quarter of the world 's population, 1.7 billion people, is obese, and the number of obese people is increasing, and the number of obesity - related deaths is rapidly increasing. In the National Nutrition Health Survey, 32.7% (33.1% in male, 32.25% in female) of the total population in Korea were obese in 2007, indicating the severity of obesity (Kim et al., 2009 ).

Obesity is an increase in the mass of adipose tissue, which includes both hyperplasia, an increase in the number of adipose tissue cells, and hypertrophy, an increase in cell size (Couillard, , C. et al., 2000). Differentiation of adipocytes involves differentiation of precursor cells (preadipocytes) into fully mature 3T3-L1 cells (matured adipocytes), leading to changes in cell morphology, gene and protein expression, and changes in hormone sensitivity. The expression of adipocytes in the adipocytes was assessed by the expression of C / EBPs (CCAAT / enhancer binding proteins), PPAR-γ (peroxisome proliferator activated receptor-γ) and SREBP-1 (sterol regulatory element-binding protein-1) , And the transcription factors induce mutual transcription and differentiate adipocytes to produce energy accumulation, fat synthesis, fat-transporting factors, thereby leading to energy production and obesity. Especially, PPAR-γ and C / EBPs inducing adipocyte-specific genes including aP2 / FABP4 (adipocyte protein 2 / fatty acid binding protein 4) and adiponectin (Kang, BT et al., 2014; Rosen, ED et al., 2001).

In the 3T3-L1 cells, adipsin, C / EBPα, C / EBPβ, aP2 / FABP4, SREBP-1c and PPAR-γ were the early differentiation transcription factors that are indicators of obesity. These include TGF-beta 1 (transforming growth factor-beta 1) and UCP-2 (uncoupling protein-2).

To treat obesity, dietary therapy, exercise therapy, behavior modification therapy that corrects wrong eating habits and lifestyle, surgery to reduce the intestine or stomach volume in surgery, appetite suppressant, diuretic, diarrhea, or fiber to give fullness And the like are being provided. However, the methods provided so far do not exert their effects or cause many side effects. In particular, sibutramine, a type of appetite suppressant, can cause side effects such as headache, dry mouth (insatiable thirst), insomnia, and constipation, and blood pressure and pulse rate may increase slightly. You have to check. In addition, orlistat, a kind of lipolytic enzyme inhibitor, has disadvantages such as frequent bowel movements and fatty changes, and a decrease in the rate of absorption of fat-soluble vitamins in the intestines due to a decrease in fat absorption rate (Morrison, RF et al., 2000).

Hyperlipidemia refers to a state in which the amount of lipid components including cholesterol and triglycerides in the blood is increased more than necessary. The cause of hyperlipidemia is due to genetic factors that increase the specific lipid in the blood and cause hyperlipemia, but it can also be caused by environmental factors such as obesity, diabetes, alcohol, stress and smoking. In addition, although hyperlipidemia itself does not indicate symptoms, it may increase the risk of coronary artery disease such as arteriosclerosis or myocardial infarction, and may cause cerebrovascular disease, peripheral vascular occlusion, and the like (Falk, E. et al., 1995).

Methods for treating hyperlipidemia include diet control, lifestyle improvement through exercise, proper weight maintenance, drug therapy, and the like. Particularly, statin drugs are widely used for drug therapy. This drug acts as an inhibitor of HMG-CoA reductase (3-hydroxy-3-methylglutaryl-coenzyme A reductase) inhibiting the synthesis of cholesterol (Low density lipoprotein cholesterol, LDL cholesterol) and drop in the blood concentration of the triglyceride is also known to have some effect, but may cause myopathy or muscle pain. Niacin, another well-known drug for the treatment of hyperlipidemia, lowers blood triglycerides and low density lipoprotein cholesterol and boosts high density lipoprotein cholesterol (HDL cholesterol) ), Liver dysfunction, and glucose control disorder. Therefore, there is a tendency to study the use of natural products having proven effectiveness and safety while minimizing such side effects.

Yard lime (Elaeagnus multiflora Thunb.) Belongs to elaeagnaceae (Elaeagnaceae) lime hawthorn (Elaeagnus), the local and distributed in the wild or cultivated as an ornamental or fruit trees (jomu Row, 1989). The fruit of the garden borealis is a pointy nucleus, long elliptical, 1.5㎝ long, ripened in June to July, and has a sweet, sweet and sour taste (Hong, JY et al., 2006; In addition, it is known that it not only eliminates the odor and eliminates the burning and bleeding, but also stops the diarrhea and bleeding, and is known to be effective for dyspepsia, osteomyelitis, edema, and impotence of menstruation (Kim, 1985; In the private sector, garden bamboo trees are sometimes called "woody summer", "lobule", "daifaho", and "lepidoptera".

Korean Patent No. 780893 discloses that a composition containing an extract of a garden fruit juice as an active ingredient has antioxidant, anti-inflammatory and whitening activity effects. In addition, the anti-oxidative and cancer cell proliferation inhibitory effects of the extracts of Y. barley juice were investigated in Hela cells (HeLa cell), MCF-7 cells (breast cancer cells) and SNU-638 cells (gastric cancer cells) (Kim, SA et al., 2007.), there is also a precedent that discloses that the extract of Y. barki extract is effective for anti-platelet aggregation and anti-inflammatory activity (Chang, ZQ et al. , 2006). However, the therapeutic effect of the barnyberry extract of the present invention on obesity or hyperlipidemia has not yet been disclosed.

Korean Registered Patent No. 780893 (antioxidant, anti-inflammatory and whitening composition containing gardenia fruit extract as an active ingredient, registered on Nov. 23, 2007)

Ko Kyung-sik, Korean Botanical Search Book, Acadamia Book, 224, 1991. Kim, Sam - Sik, The original color picture book of Korea, Keimyungsa, 403, 1986. Kim, Hye - Ryeon, et al., A Study on Linkage and Activation of National Obesity Project for Children and Adolescents, Ministry of Health, Welfare and Family Affairs, 2009. I, et al., Clinical Obesity Pharmacology, Hanmi Medical, 2009. Park Jong - hee, Korean herbal book, book publishing Shinil Corporation, 953-954, 2004. Lee, Chang - Bok, The Korean Botanical Illustrated Book, The. Chung Moo-ryou, Korean-color illustrations, Academy, 372, 1989. Chang, Z. Q. et al., Anti-platelet aggregation and anti-inflammatory activity for extracts of Elaeagnus multiflora, Korean Journal of crop Science, 516-517, 2006. Couillard, C. et al., Hyperleptinemia is more closely associated with adipose cell hypertrophy than with adipose tissue hyperplasia, Int. Journal. Obes. Relat. Metab. Disord., 24, 782-788,2000. Falk, E. et al., Coronary plaque disruption, Circulation, 92 (3), 657-671, 1995. Homma, H. et al., Estrogen suppresses transcription of lipoprotein lipase gene, Journal. Biol. Chem., 275, 11404-11411, 2000. Hong, J. Y. et al., Anti-oxidant Activity of Ethanolic Extracts from Fruits of Elaeagnus multiflora Thunb. during Maturation, Korean Journal of Food Preserv., 13 (5), 643-648, 2006. Kang, B. T. et al., Effects of steamed Polygonatum odoratum extract on inhibition of adipocyte differentiation and lowering lipidis 3T3-L1 adipocytes, Korean Journal of Herbology, 29 (2), 15-21, 2014. Kim, S. A. et al., Antioxidative and Cytotoxic Effects of Solvent Fractions from Elaeagnus multiflora, Korean Journal of Food and Nutrition, 20 (2), 134-142, 2007. Morrison, R. F. et al., Hormonal signaling and transcriptional control of adipocyte differentiation, J. Nutr., 130 (12), 3116-3121, 2000. Montagne, A. et al., Quantification of ovine cytokine gene expression by a competitive RT-PCR method, J. Immunol. Methods., 253 (1-2), 83-93, 2001. Munro, J. F. et al., Comparison of continuous and intermittent anorectic therapy in obesity, Br. Med. J., 1 (5588), 352-354, 1968. Park, Y. W., Efficacy and Safety of Phentermine for Obese Patients: A Preliminary Open-Label Study, Korean Journal of Obesity, 14 (1), 1-8, 2005. Rosen, E. D. et al., Molecular regulation of adipogenesis, Annu. Rev. Cell. Dev. Biol., 16, 145-171, 2001. Stephen, J. G., Allergy, Current Biology, 10 (3), 93-95, 2000. Weintraub, M. et al., Long-term weight control study. I (weeks 0 to 34). The enhancement of behavior modification, caloric restriction, and exercise by fenfluramine plus phentermine versus placebo, Clin. Pharmacol. Ther., 51 (5), 586-594, 1992.

It is an object of the present invention to provide a composition for prevention or treatment of obesity or hyperlipidemia, which comprises extract of barnyardgrass. More particularly, the present invention relates to a method for preventing or preventing obesity or hyperlipidemia, which comprises extracting a gardenia extract having an effect of inhibiting lipid differentiation and fat accumulation in adipocytes and reducing the level of total cholesterol or low density lipoprotein cholesterol in serum, And to provide a therapeutic composition.

The present invention relates to a composition for preventing or treating obesity or hyperlipidemia, which comprises Elaeagnus multiflora Thunb. Extract.

The gardenia extract can be extracted with water, C1 to C4 lower alcohols, or a mixed solvent thereof.

The gardenia extract may be extracted with a mixture of C1 to C4 lower alcohols or water and C1 to C4 lower alcohols.

The composition may be formulated into a pharmaceutical dosage form with the addition of a pharmaceutically acceptable carrier, excipient or diluent.

In another aspect, the present invention relates to a health functional food for preventing or ameliorating obesity or hyperlipidemia, which comprises an extract of a garden lotus extract.

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition for preventing or treating obesity or hyperlipidemia, which comprises extracts of barnyardgrass, wherein said barnyardgrass extract is obtained by extracting with water, C1 to C4 lower alcohols or a mixed solvent thereof, and said C1 The alcohols to C4 may be methanol, ethanol, propanol, isopropanol, butanol, and the like. Preferably, the gardenia extract is an extract obtained by extracting gardenia juice with a C1 to C4 lower alcohol or a mixed solvent of water and C1 to C4 lower alcohol, more preferably a 30 to 100% aqueous ethanol solution (or ethanol ), And most preferably, the extract is extracted with an aqueous 50 to 80% ethanol solution.

The yam is preferably a fruit, a stem, a leaf, a branch, a root, or the like, preferably a fruit.

On the other hand, the gardenia extract of the present invention may be used as it is or may be prepared into powder by freeze-drying or spray-drying.

In addition, the present invention provides a pharmaceutical composition for preventing or treating obesity or hyperlipidemia, which comprises extract of barnyardgrass. The pharmaceutical composition containing the extract may be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method Can be used. Examples of carriers, excipients and diluents that can be contained in the pharmaceutical 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. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant 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 or 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. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The dosage of the pharmaceutical composition comprising the gardenia extract of the present invention will depend on the age, sex, body weight, the specific disease or condition to be treated, the severity of the disease or condition, the route of administration, and the judgment of the prescriber. Dosage determinations based on these factors are within the level of ordinary skill in the art and generally the dosage ranges from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferable dosage is 1 mg / kg / day to 500 mg / kg / 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 pharmaceutical composition comprising the gardenia extract of the present invention can be administered to mammals such as rats, livestock, and humans in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. Since the extract of the present invention has little toxicity and side effects, it can be safely used even for long-term administration for the purpose of prevention.

The present invention also provides a health functional food for preventing or ameliorating obesity or hyperlipidemia, comprising a barnyard extract and a pharmaceutically acceptable food-aid additive. The extract may be added to the health functional food of the present invention in an amount of 0.001 to 100% by weight. The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids. Examples of the foods to which the extract of the present invention can be added include various foods, beverages, gums, tea, vitamins .

The present invention relates to a composition for the prevention or treatment of obesity or hyperlipidemia, which comprises extract of barnyardgrass. The extract of yam is excellent in the effect of inhibiting lipid differentiation and fat accumulation in adipocytes and reducing the level of total cholesterol or low density lipoprotein cholesterol in serum and is useful as a composition for preventing or treating obesity or hyperlipemia .

FIG. 1 shows the results of an MTT assay for confirming the cell viability of the aqueous extract solution of 80% [v / v] ethanol of the present invention.
FIG. 2 shows the results of Oil-Red-O staining showing the effect of reducing the number of lipid droplets in 3T3-L1 cells in which the extract of aqueous ethanol solution of 80% [v / v] ethanol of the present invention was induced to differentiate into adipocytes.
FIG. 3 is a graph showing that in the 3T3-L1 cells in which the extract of aqueous ethanol solution of 80% [v / v] ethanol of the present invention was induced to differentiate into adipocytes, adipocyne (FIG. 3A), C / EBPα ), RQ (relative to real-time PCR) indicating that mRNA expression of C / EBP? (FIG. 3C), aP2 / FABP4 (FIG. 3D), SREBP-1c (FIG. 3E) and PPAR- quantitative analysis.
FIG. 4 shows that TGF-β1 (FIG. 4A) and UCP-2 (FIG. 4B), which are differentiation-inhibiting transcription factors, were detected in 3T3-L1 cells in which the aqueous solution of 80% [v / v] ethanol aqueous solution of the present invention was induced to differentiate into adipocytes. ) Relative to that of the control group.
Figure 5 shows that the aqueous solution of aqueous ethanol 80% [v / v] of the present invention in the obesity animal model C57bl / 6 mouse has the effect of reducing weight loss (Fig. 5A) and reducing dietary efficiency levels (Fig. 5B) Graph.
FIG. 6 shows that the extract of aqueous ethanol solution of 80% [v / v] ethanol of the present invention of the present invention had a significant effect on serum triglyceride (FIG. 6A), free fatty acid (FIG. 6B), total cholesterol (FIG. 6C) And low-density lipoprotein cholesterol (Fig. 6D).
FIG. 7 is a graph showing that the extract of aqueous ethanol solution of 80% [v / v] ethanol of the present invention has an effect of reducing serum leptin in obese animal model C57bl / 6 mouse.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, the intention is to provide an exhaustive, complete, and complete disclosure of the principles of the invention to those skilled in the art.

&Lt; Example 1: Preparation of Gardenia extract &

Example 1-1. Preparation of aqueous extracts of 80% [v / v] ethanol aqueous solution of yam

The fruit of Elaeagnus multiflora Thunb. Used in the present invention was collected in June 2014 in the area of Misan-myeon, Boryeong-si, Chungcheongnam-do. 200 g of the above-mentioned barnyardgrass was mixed with 1.5 L of an 80% [v / v] ethanol aqueous solution, sonicated for 30 minutes, filtered under reduced pressure through a 3 mm filter paper (Whatman, Maidstone, England) To obtain an aqueous solution. The filtered aqueous solution of 80% [v / v] ethanol was concentrated using a rotary evaporator (Eyela, Japan) and then lyophilized to obtain an aqueous solution of 80% [v / v] ethanol aqueous solution in powder form And dissolved and stored at a concentration of 200 mg / ml in tertiary distilled water.

Examples 1-2. Preparation of 30% [v / v] ethanol aqueous solution extract of garden yard

Except that 30% [v / v] ethanol aqueous solution extract of yam boreal water [v / v] was prepared by using a 30% [v / v] ethanol aqueous solution instead of the 80% [v / v] ethanol aqueous solution. .

Examples 1-3. Preparation of 50% [v / v] ethanol aqueous solution extract of garden yard

A vinegar extract was prepared in the same manner as in Example 1-1 except that 50% [v / v] ethanol aqueous solution extract of vinegar water [v / v] was used instead of 80% [v / v] .

Examples 1-4. Preparation of 100% Ethanol Extract of Gardenia L.

100% ethanol extract of yam borax was prepared by using 100% ethanol instead of 80% [v / v] ethanol aqueous solution.

Examples 1-5. Preparation of water extract of gardenia

200 g of yam borealis fruit was mixed with 2 ℓ of water, extracted at 90 캜 for 10 hours, and then filtered under reduced pressure with a 3 mm filter paper to obtain a water extract of yam water. The water extract was lyophilized to prepare a water extract of Y. pombeii.

&Lt; Example 2: Measurement of cell viability &

In order to measure the cell viability of the barnyardgrass extract prepared in Example 1 of the present invention, MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay was performed.

First, the frequency division by 100㎕ in 3T3-L1 cells ^{(American Type Culture Collection, ATCC ®} , CL-173) concentration of 1 × 10 ⁶ cells / ㎖ to a 96-well plate (96well microtiter plate) and incubated for 24 hours. The extracts of barnyardgrass prepared in Examples 1-1 to 1-5 were added to the medium of FBS (Fetal Bovine Serum, Hyclone, USA) and DMEM without antibiotics (Dulbecco's Modified Eagle's Medium, Sigma, USA) Ml, and incubated with 3T3-L1 cells for 24 to 72 hours. Then, 0.5 mg / ml of MTT solution was added, and the cells were further cultured for 4 hours. After completion of the reaction, the supernatant was removed and dried in a dark place for 30 minutes. Then, 100 μl of DMSO (dimethyl sulfoxide) was added thereto and reacted for 1 hour. Then, the absorbance was measured at 540 nm using an absorbance meter (ELISA reader, molecular device, USA), and the cell viability of the following formula 1 was calculated and shown in FIG.

[Equation 1]

As a result of the MTT assay shown in Fig. 1, the group treated with the extract of aqueous ethanol solution of 80% [v / v] yam in Example 1-1 of the present invention had a cell survival rate similar to that of the control (0 / / , Indicating that cytotoxicity does not appear. In addition, although not shown in FIG. 1, the extracts of aqueous ethanol solution of 30-50% [v / v], 100% ethanol extract and water extract of barnyardgrass of Examples 1-2 to 1-5 were found not to be cytotoxic.

&Lt; Example 3: Identification of lipid differentiation and fat accumulation in adipocytes >

In order to investigate the effects of extracts from the garden liquor of Examples 1-1 to 1-5 on lipid differentiation and fat accumulation in 3T3-L1 cells, the lipid droplets were specifically reacted, (2.5-dimethyl-4- (2-5-dimethylphenyl) phenyldiazenyl) azonapthalen-2-ol, which can be visualized, was stained (Homma, H. et al ., 2000).

3T3-L1 cells were cultured in a CO ₂ incubator (37 ° C, 5%) using DMEM medium containing 10% [v / v] FBS, 1% [w / v] penicillin G- sodium and streptomycin sulfate. CO ₂ ) and subcultured at 3-day intervals. When 3T3-L1 cells reached the confluent stage by inoculating 3T3-L1 cells (preadipocytes) in the cultured undifferentiated state with the same number (2 x 10 ⁴ ) in a 24-well plate, Differentiation induction medium and each sample were treated for 48 hours to induce differentiation into adipocytes. For the first 48 hours, 3T3-L1 cell cultures were treated with 0.25 μM dexamethasone, 0.5 μM IBMX (3-isobutyl-1-methylxanthine) and 10 μg / After that time, only 5 μg / ml of insulin was added to the 3T3-L1 cell culture medium. As a control group, 17 [beta] -estradiol (E2), which is known to reduce PPAR-gamma induced adipocyte differentiation and adipocyte differentiation gene expression, was treated with 10 [mu] M, and in the yard of Examples 1-1 to 1-5 of the present invention The extracts of Bombyx mori were treated with 200 ㎍ / ㎖, 100 ㎍ / ㎖ and 10 ㎍ / ㎖, respectively. Then, the culture of each 3T3-L1 cell was removed, washed with PBS (phosphate buffered saline), and then fixed with 10% [v / v] formaldehyde solution for 1 hour or more. After washing with PBS and adding Oil-Red-O solution, the mixture was stained for 30 minutes at room temperature, washed with 70% [v / v] ethanol aqueous solution, and observed for lipid accumulation under a microscope. The results are shown in Table 1 and FIG. 2 .

Condition Number of fat particles Example 1-1 Vinegar 80% [v / v] ethanol aqueous solution extract 200 [mu] g / ml 52 100 mu g / ml 57 10 mu g / ml 65 Examples 1-2 Vinegar 30% [v / v] ethanol aqueous solution extract 200 [mu] g / ml 68 100 mu g / ml 74 10 mu g / ml 83 Example 1-3 50% vinegar [v / v] aqueous solution of ethanol 200 [mu] g / ml 60 100 mu g / ml 65 10 mu g / ml 77 Examples 1-4 100% ethanol extract of garden water 200 [mu] g / ml 66 100 mu g / ml 72 10 mu g / ml 81 Examples 1-5 Gardenia water extract 200 [mu] g / ml 85 100 mu g / ml 91 10 mu g / ml 93

Table 1 and Fig. 2A show the result of numerical representation of the results of Fig. 2B. In the group induced by differentiation of 3T3-L1 cells using differentiation-inducing medium, the formation of cytoplasmic lipids was actively induced, whereas in the 17β-estradiol-treated group, the number of lipid droplets decreased . In the case of the group treated with the extract of Example 1-1 of the present invention, it was confirmed that the effect of inhibiting lipid differentiation and fat accumulation in adipocytes was remarkably excellent due to a decrease in the number of lipids in a concentration-dependent manner.

&Lt; Example 4: Expression analysis of adipocyte differentiation gene and differentiation inhibitory gene >

C / EBP?, AP2 /?,? /?,?,?,?,?, And?) In the 3T3-L1 cells of the aqueous ethanol solution of 80% FABP4, SREBP-1c and PPAR-γ) and mRNA expression of differentiation-inhibiting transcription factors (TGF-β1 and UCP-2)

Example 4-1. RNA isolation from 3T3-L1 cells

To extract mRNA, the undifferentiated 3T3-L1 cells were transfected with RNAzol B 500 μl of the reagent was added and pulverized with a homogenizer until it was dissolved. 50 μl of chloroform was added to the mixed suspension, mixed for 15 seconds, left on ice for 15 minutes, and centrifuged at 13,000 rpm to recover about 200 μl of supernatant. To the supernatant was added 200 [mu] l of 2-propanol (propanol), mixed slowly and left on ice for 15 minutes. Thereafter, the cells were centrifuged at 13,000 rpm, washed with an 80% [v / v] ethanol aqueous solution, and then dried in a vacuum pump for 3 minutes to extract RNA. The RNA was dissolved in 20 μl of DEPC (diethyl pyrocarbonate) -treated water and used for cDNA synthesis.

Example 4-2. Reverse transcription polymerase chain reaction (PCR)

For the reverse transcription reaction, 2 μg of total RNA and 2U of DNase I (deoxyribonuclease I, 10 U / ml) were reacted at 37 ° C for 30 minutes and then denatured by heating at 75 ° C for 10 minutes. Thereafter, a mixture of 2.5 μl 10 mM dNTPs, 1 μl random sequence hexanucleotides (25 pM / 25 μl), 1 μl RNase inhibitor (20 U / ml), 1 μl 100 mM DTT (dithiothreitol), 4.5 μl 5 × RT buffer (250 mM pH 8.3 Tris _{-HCl, 375mM KCl, 15mM MgCl 2} ) , and then added, was added M-MLV RT (moloney-murine leukemia virus reverse transcriptase, 200U / ㎕) 1㎕ of the final volume by the addition of DEPC-treated distilled water, so that the 20㎕ Respectively. The reaction mixture was mixed well, centrifuged at 2,000 rpm for 5 seconds, and incubated at 37 ° C for 60 minutes to synthesize cDNA. Then, the reaction mixture was allowed to stand at 95 ° C for 5 minutes to inactivate M-MLV RT, cDNA was used for PCR (Montagne, A. et al., 2001).

Example 4-3. Real time polymerase chain reaction (PCR)

The cDNA synthesized in Example 4-2 was subjected to real-time PCR using a 7500 real-time PCR system (Applied Biosystems, USA) (Stephen, J. G., 2000). The primers used for real-time PCR amplification were oligonucleotides for SYBR of adipocin, C / EBPα, C / EBPβ, SREBP-1c, aP2 / FABP4, PPAR-γ, TGF-β1 and UCP- (G3PDH probe) (Glyceraldehyde-3-phosphate dehydrogenase probe, Applied Biosystems) was used as a control group. The nucleotide sequences of these were shown in Table 2.

Gene Primer Sequence Adipine Forward 5'-CACCATCGACCACGACCTC-3 ' Reverse 5'-AGTGTGGCCTTCTCCGACAG-3 ' C / EBPα Forward 5'-TGGACAAGAACAGCAACGAGTAC-3 ' Reverse 5'-CGGTCATTGTCACTGGTCAACT-3 ' C / EBP? Forward 5'-AAGCTGAGCGACGAGTACAAGA-3 ' Reverse 5'-GTCAGCTCCAGCACCTTGTG-3 ' aP2 / FABP4 Forward 5'-TGGGAACCTGGAAGCTTGTCTC-3 ' Reverse 5'-GAATTCCACGCCCAGTTTGA-3 ' SREBP-1c Forward 5'-AGCCTGGCCATCTGTGAGAA-3 ' Reverse 5'-CAGACTGGTACGGGCCACAA-3 ' PPAR-γ Probe-FAM 5'-TCGGAATCAGCTCTGTGGACCTCTCC-3 ' TGF-β1 Forward 5'-CCCGAACTGTGACTTTTGCT-3 ' Reverse 5'-CCTCTGGATAGCGAGGACTG-3 ' UCP-2 Forward 5'-TTCAAATGAGATTGTGGGAAAAT-3 ' Reverse 5'-ACCGATACAGTACAGTACAGTA-3 '

As a reaction solution, a mixture of TaqMan PCR master and SYBR Green PCR master was used and the final concentration of the primer was 200 nM. Real-time PCR was performed at 50 < 0 > C 2 min-1 cycles; 94 ° C 10 min -1 cycle; And (40 cycles of 95 ° C for 9 seconds and 60 ° C for 1 minute). The results are shown in FIGS. 3 and 4.

3, in the 3T3-L1 cells in which the extract of aqueous ethanol solution of 80% [v / v] ethanol of the present invention was induced to differentiate into adipocytes, adipocin (FIG. 3A), C / EBPα (FIG. 3B), C / EBP (FIG. 3C), aP2 / FABP4 (FIG. 3D), SREBP-1c (FIG. 3E) and PPAR-γ When 3T3-L1 cells were treated at a concentration of 200 μg / ml, the effect was equivalent to or higher than that of 17β-estradiol as a control group.

4, in the 3T3-L1 cells in which the extract of aqueous ethanol solution of 80% [v / v] ethanol of the present invention was induced to differentiate into adipocytes, TGF-β1 (FIG. 4A) -2 (Fig. 4B).

&Lt; Example 5: Analysis of obesity effect in animal model >

Example 5-1. Weight change and dietary efficiency analysis

① Obesity induction and weight change analysis of animal model C57bl / 6 mouse

For this experiment, 7 week old male C57bl / 6 mice were randomly assigned to the laboratory environment and weighed 23 ± 1g. Weighing 230 ± (D ) &Lt; / RTI > 270 x (H) 220 mm). The breeding environment provided an SPF environment with constant temperature (22 ± 2 ° C), constant humidity (50 ± 5%), light intensity on 12 hour intervals (light on 07: 00 ~ 19: 00) It was freely supplied. The subjects were fed a high fat diet (HFD, # D12492, Rodent Diet with 60% kcal% fat, Research Diets, New Brunswick, USA) for 2 weeks on an 8 week old C57bl / Was reached between 28 and 29g.

10-week-old obese animal model C57bl / 6 mice between the average weights of 28-29 g were treated with high-fat diet and 0.2 ml of each sample twice a day for 5 weeks. The comparative groups include xenical (containing orlistat, which blocks the decomposition and absorption of triglycerides through the mechanism of reversible blocking of pancreatic lipase, resulting in a decrease in calories absorbed into the small intestine) and conjugated linoleic acid Inhibition of lipoprotein lipase inhibits lipid accumulation in peripheral tissues and thus contributes to the synthesis of adipose tissue (Munro, JF et al., 1968; Park, YW, 2005; Weintraub, M. et al., 1992; Na, et al., 2009).

Each experimental mouse group was fed with only basic diet (only the basic diet was fed to normal mice), high-fat diet, high fat diet and 15.6 mg / kg, high fat diet and conjugated linoleic acid 100 mg / ㎏, and a group treated with high - fat diet and 50 ㎎ / ㎏ or 200 ㎎ / ㎏ of 80% [v / v] ethanol aqueous solution extract. After 5 weeks of breeding, the animals were fasted for 6 hours, anesthetized with anesthetic, and blood and organs were detected to measure weight change and dietary efficiency. The results are shown in FIGS. 5A and 5B.

FIG. 5A is a graph showing the mouse weight change level of the above six groups. In the group that consumes only the high fat diet, the body weight increases by 32.5% or more as compared with the group that only consumes the basic diet, , And 21.1% and 17.3%, respectively, in the group administered with the high fat diet and the conjugated linoleic acid and the high fat diet group and the 200 mg / kg of the aqueous ethanol solution of 80% [v / v] %, And 12.3%, respectively.

② Dietary efficiency analysis

The body weight and food intake of the six groups of obese animal models C57bl / 6 mice treated with the samples for 5 weeks were measured and recorded at 9:00 on Wednesdays, and the feeding efficiency (% Is calculated by dividing the total weight gain (g) by the total feed intake (g) by substituting the following equation (2) (the smaller the number is, the more the effect of controlling obesity is shown in FIG. 5B).

[Equation 2]

As shown in FIG. 5B, about 48.8% of the high-fat diet and high fat diet group and about 23.4% of the high fat diet group and the conjugated linoleic acid diet group had higher FER than the high fat diet group, And 200mg / kg of ethanol extract of barnyardgrass decreased to 23.3%. It can be seen that the extract of aqueous solution of 80% [v / v] ethanol in the yam has excellent weight loss effect.

Example 5-2. Serum mass analysis

Blood biochemical tests were performed by collecting blood from an obese animal model C57bl / 6 mouse of the 6 groups of Example 5-1 in which the samples were treated for 5 weeks. Each blood was centrifuged at 3,000 rpm and 4 ° C for 15 minutes, and the serum was collected and stored at -70 ° C. Within 30 minutes after the blood collection, the serum was used to measure the amount of triglyceride free lipid, triglyceride, free fatty acid, total cholesterol and lower-density lipoprotein cholesterol were measured using a biochemical automatic analyzer (Hitachi-720, Hitachi Medical, Japan) The results are shown in Fig.

6, the extract of aqueous ethanol solution of 80% [v / v] ethanol of the present invention was administered to 6 groups of obese animal model C57bl / 6 mice of Example 5-1, (FIG. 6B), total cholesterol (FIG. 6C) and low density lipoprotein cholesterol (FIG. 6D).

On the other hand, in the same manner as in Example 5-1, the aqueous extracts of 30-80% [v / v] ethanol aqueous solution, 100% ethanol extract of yam and 100% The model C57bl / 6 mice were treated to compare the levels of total cholesterol and LDL-cholesterol in the mice, and the results are shown in Table 3 below.

Condition Serum total cholesterol (㎎ / ㎗) Low-density lipoprotein cholesterol (㎎ / ㎗) Example 1-1 Vinegar 80% [v / v] ethanol aqueous solution extract 200 [mu] g / ml 162 11.2 50 mu g / ml 174 14.3 Examples 1-2 Vinegar 30% [v / v] ethanol aqueous solution extract 200 [mu] g / ml 175 13.7 50 mu g / ml 206 16.8 Example 1-3 50% vinegar [v / v] aqueous solution of ethanol 200 [mu] g / ml 166 11.6 50 mu g / ml 183 15.2 Examples 1-4 Vinegar 100% [v / v] ethanol extract 200 [mu] g / ml 173 13.6 50 mu g / ml 188 16.4 Examples 1-5 Gardenia water extract 200 [mu] g / ml 211 18.2 50 mu g / ml 228 19.6

Example 5-3. Analysis of serum hormone protein

Serum was separated from the experimental animals in which the 6 groups of Example 5-1 were administered for 5 weeks, and the level of leptin (hormone protein for maintaining constant body fat secreted from adipose tissue) in the serum was assayed Respectively. For this, ELISA was performed using an enzyme-linked immunosorbent assay kit (R & D System, USA). 100 μl of the coating antibody was dispensed into each microwell according to the manufacturer's instructions, reacted at 4 ° C for 16 hours, and each well was washed with a wash buffer. Then, 200 μl of assay diluent was added and the wells were sealed for 1 hour. After reacting at room temperature, the microplate was washed and the reaction solution (SYBR PCR Master mix, ABI, USA) was prepared 100 μl each was added to each well, and the wells were incubated for 1 hour, followed by reaction at room temperature. Subsequently, the microplate was washed and a substrate solution was prepared. 100 μl of the substrate solution was added to each well. After 30 minutes of reaction at room temperature in a dark place, 50 μl of stop solution was added to each well, The absorbance was measured at 450 nm using a microplate spectrophotometer and is shown in FIG.

7, the extract of the aqueous ethanol solution of 80% [v / v] ethanol of the present invention had excellent effect of reducing the serum leptin level in the C57bl / 6 mouse of the obesity animal model of the 6 groups of Example 5-1 .

&Lt; Formulation Example 1 >

Formulation Example 1-1. Manufacture of tablets

200 g of an aqueous 80% [v / v] ethanol aqueous solution of Example 1-1 of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. To this mixture was added a 10% gelatin solution, which was pulverized and passed through a 14-mesh sieve. This was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into tablets.

Formulation Example 1-2. Injection preparation

1 g of an aqueous solution of 80% [v / v] aqueous ethanol of the present invention of Example 1-1, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20 DEG C for 30 minutes.

<Formulation Example 2: Food Preparation>

Formulation Example 2-1. Manufacture of cooking seasonings

A vegetable soup for health promotion was prepared by adding 1% by weight of an aqueous solution of 80% [v / v] aqueous ethanol solution of Example 1-1 of the present invention to a cooking seasoning.

Formulation Example 2-2. Manufacture of flour food products

The bread, cake, cookies, crackers and noodles were prepared by adding 0.1% by weight of the extract of aqueous solution of 80% [v / v] ethanol of Example 1 of the present invention to wheat flour, .

Preparation Example 2-3. Manufacture of soups and gravies

The health enhancing soup and the juice were prepared by adding an aqueous solution of 80% [v / v] aqueous ethanol solution of Example 1 of the present invention to 0.1% by weight of soup and juice.

Formulation Example 2-4. Manufacture of dairy products

An aqueous solution of 80% [v / v] aqueous ethanol solution of Example 1-1 of the present invention was added to milk in an amount of 0.1% by weight, and various dairy products such as butter and ice cream were prepared using the milk.

Formulation Example 2-5. Vegetable juice manufacturing

A vegetable juice for health promotion was prepared by adding 0.5 g of an aqueous solution of 80% [v / v] aqueous ethanol solution of Example 1-1 of the present invention to 1,000 ml of tomato juice or carrot juice.

Formulation example 2-6. Fruit juice Produce

The fruit juice for health promotion was prepared by adding 0.1 g of the aqueous solution of 80% [v / v] aqueous ethanol solution of Example 1-1 of the present invention to 1,000 ml of apple juice or grape juice.

Claims

A composition for preventing or treating obesity or hyperlipidemia, which comprises an extract of Elaeagnus multiflora Thunb.

The method according to claim 1,
The composition for preventing or treating obesity or hyperlipidemia according to claim 1, wherein the yam extract is obtained by extracting yam water with water, C1-C4 lower alcohol, or a mixed solvent thereof.

3. The method of claim 2,
Wherein said yam extract is obtained by extracting yam bore water with a C1 to C4 lower alcohol or a mixed solvent of water and C1 to C4 lower alcohols.

The method according to claim 1,
A composition for preventing or treating obesity or hyperlipidemia, wherein the composition is formulated into a pharmaceutical dosage form by adding a pharmaceutically acceptable carrier, excipient or diluent.

A health functional food for preventing or ameliorating obesity or hyperlipidemia, which comprises an extract of Elaeagnus multiflora Thunb.

6. The method of claim 5,
Wherein the yam extract is obtained by extracting yam bore water with water, C1 to C4 lower alcohols, or a mixed solvent thereof, to thereby prevent or ameliorate obesity or hyperlipidemia.

The method according to claim 6,
Wherein the yam water extract is obtained by extracting yam bore water with a C1 to C4 lower alcohol or a mixed solvent of water and a C1 to C4 lower alcohol, to thereby prevent or ameliorate obesity or hyperlipidemia.