KR20140045134A - Pharmaceutical composition for preventing or treating obesity or metabolic disorders comprising aster glehni extract as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising an Aster glehni extract or a fraction thereof as an active ingredient for preventing or treating obesity or metabolic diseases and a functional health food for preventing or treating obesity or metabolic diseases. The Aster hlehni extract or the fraction thereof, according to the present invention, reduces weight, an amount of fat in a body, glucose and total cholesterol levels in serum, an amount of accumulated fat in a body (liver and intestinal adipose tissues), and especially an expression of genes that are engaged in metabolizing fat and forming adipocytes, and thus can be extremely useful in preventing or treating obesity or metabolic diseases.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating obesity or metabolic diseases, which comprises an extract of Aspergillus oryzae as an active ingredient,

The present invention relates to a method of screening for Aster The present invention relates to a pharmaceutical composition for preventing or treating obesity or a metabolic disease containing an extract or a fraction thereof as an active ingredient and a health functional food for preventing or ameliorating obesity or metabolic diseases.

Overweight and obesity are biological phenomena caused by the interaction of complex, genetic, metabolic, environmental, and behavioral factors, defined as the accumulation of abnormal or excessive fat, and can have adverse health effects. Medically, it is classified as obesity if the body mass index (BMI) is 30 or more (ie, 30% or more of the standard body weight) or if the BMI is 27 or more. In addition, obesity is classified as a disease associated with other circulatory diseases such as diabetes (especially type 2 diabetes), hypertension, and hyperlipemia. In particular, obesity is known to be an important factor causing various adult diseases such as hypertension, type 2 diabetes, cancer, gallbladder disease, hyperlipidemia, arteriosclerosis and the like. The causes of obesity known to date are known to be over 70% of genetic predisposition, and other environmental factors include high-fat diet intake and lack of exercise. However, recently, the imbalance between the amount of energy consumed and the amount of energy consumed has caused obesity Is emerging. In other words, it is difficult to see it as a genetic cause because the incidence has rapidly increased even though the genetic predisposition has not changed much in the meantime. Therefore, the perception that the genetic and environmental factors that destroy the energy balance are important factors of obesity spread have. At least 28 million adults die each year as a result of obesity.

Fat stored in fat cells is used as an important energy source in the body. However, as the obesity progresses, not only the number of adipocytes increases in number but also a large amount of triglyceride synthesis due to the differentiation of excessive adipocytes is accompanied by a morphological change including an increase in adipocyte size and a change in gene expression. The increase in fat cell size is induced by the synthesis and storage of surplus energy in the form of triglycerides. On the other hand, according to the storage of fat, the increase in the size of adipocytes can increase its diameter to about 20 times, and as a result, the cell volume is increased to several thousand times. However, since the differentiation of new precursor adipocytes into adipocytes is ineffective as a dietary regulator, in order to control the fundamental treatment or inhibition of obesity, the differentiation process of adipocytes It is important to adjust.

Adipocyte differentiation is stimulated by stimuli such as insulin-like growth factor-1, growth hormone and the like, in which the CCAAT enhancer-binding protein (C / EBP) family, An increase in transcription factors such as peroxisome proliferator-activated receptor (PPAR) gamma is observed. These transcription factors, along with adipocyte regulators, promote the differentiation of adipocytes and increase the expression of enzymes such as aP2, a fatty acid binding protein, and fatty acid synthase. On the other hand, it has been reported that excessive accumulation of triglycerides is involved in the progression of fatty liver [J. Clin. Invest., 98, 1575 1584 (1996)].

Currently, there are treatments such as Orlistat to suppress fat absorption and absorption of fat, fluoxetine to suppress appetite, fendimetrazine, and ephedrine, which is a metabolic accelerator. However, the above-mentioned conventional therapeutic agents have side effects such as heart diseases, respiratory diseases, nervous system diseases and the like, and the persistence of their effects is also low, and it is necessary to develop a further improved therapeutic agent for obesity.

On the other hand, Aster glehni native to Ulleungdo in Korea is about 1 m high, with fine hairs on the stem and rootstock on the side. Leaves are staggered and when the flowers are bloomed, the leaves on the bottom are blurred. The stem leaf is long and oval with a length of 1319cm and a butterfly of 46cm. There are irregular sawtooth on the edge, hairs on both sides, there is a gland point on the back, and the flower blooms in 810th month, white and 1.5cm in diameter, and runs on the main stem and the end of the branch.

The Korean Peninsula is said to have an efficacy that removes wind, detoxifies, detoxifies, removes follicles and stops coughing. It is also known to be able to cure colds, tonsillitis, bronchitis, sunburn, poisonous bites, snakebites, and bee-struck wounds due to heat. However, the anti-obesity-related activity of the scabbard is not known.

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of obesity or metabolic diseases which contains Aspergillus oryzae extract or a fraction thereof as an active ingredient.

It is another object of the present invention to provide a health functional food for preventing or ameliorating an obesity or metabolic disease which contains an extract of Scutellaria pertussis or a fraction thereof.

In order to achieve the above object, the present invention provides a method for inhibiting the expression of genes involved in body weight loss, reduction of body fat, reduction of plasma glucose and total cholesterol level, reduction of fat accumulation in the body (liver and visceral fat tissue) Or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition for preventing or treating obesity or a metabolic disease containing the extract or a fraction thereof, which can be used for prevention or treatment of obesity or metabolic diseases,

The present invention also provides a health functional food for preventing or ameliorating an obesity or metabolic disease which contains an extract of Fusarium spp. Or a fraction thereof.

The extract or its fraction according to the present invention reduces body weight, reduces body fat, decreases plasma glucose and total cholesterol levels, decreases fat accumulation in the body (liver and visceral fat tissue) Metabolism and adipocyte formation, it can be very useful for prevention or treatment of obesity or metabolic diseases.

FIG. 1 shows the result of measurement of the adipocyte differentiation effect on the differentiated adipocyte extract. FIG. 1 (A) shows a picture of the fat droplet in the differentiated adipocytes, (B) ), And (C) and (D) are results of measuring the degree of intracellular accumulation of triglyceride by the Adipo-red assay. # p < 0.05 vs. N group, ## p < 0.01 vs. N group, ### p < 0.001 vs N group. * p <0.05 vs. H group, ** p <0.01 vs H group, *** p <0.001 vs H group.
FIG. 2 is a graph showing the results of measuring the effect of the extract of Mulberry japonica on the expression of adipogenic markers. The results obtained by treating the differentiated adipocytes with 100, 200 and 400 g / ml of Mulberry japonica extract, respectively to be. # p < 0.05 vs. N group, ## p < 0.01 vs. N group, ### p < 0.001 vs N group. * p <0.05 vs. H group, ** p <0.01 vs H group, *** p <0.001 vs H group.
FIG. 3 shows the results of measurement of the effect of the extracts of Mulberry japonica on the body weight, fat pad mass and liver mass of N, HF, AG1 and AG5 group mice, (A) ) Is the degree of weight gain, (C) is the visceral fat-pad mass, and (D) is the liver mass. # p < 0.05 vs. N group, ## p < 0.01 vs. N group, ### p < 0.001 vs N group. * p <0.05 vs. H group, ** p <0.01 vs H group, *** p <0.001 vs H group.
FIG. 5 shows the results of measuring the effect of the extract of Mortalia japonica on the biochemical indicators of plasma of experimental mice. # p < 0.05 vs. N group, ## p < 0.01 vs. N group, ### p < 0.001 vs N group. * p <0.05 vs. H group, ** p <0.01 vs H group, *** p <0.001 vs H group.
FIG. 6 shows the result of measurement of the adipogenic signal genes (A) expression in the epididymal adipose tissue (Real-time PCR analysis (B) ) Is the result of Western blotting the protein levels of PPAR and C / EBP. # p < 0.05 vs. N group, ## p < 0.01 vs. N group, ### p < 0.001 vs N group. * p <0.05 vs. H group, ** p <0.01 vs H group, *** p <0.001 vs H group.

The present invention relates to a pharmaceutical composition for preventing or treating an obesity or metabolic disease containing an extract of Aster glehni or a fraction thereof as an active ingredient and a health functional food for preventing or ameliorating obesity or metabolic diseases.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a method of screening for Aster The present invention provides a pharmaceutical composition for preventing or treating obesity or a metabolic disease containing an extract or a fraction thereof as an active ingredient.

In the present invention, it is possible to use a commercially-available, commercially available or purchased or cultivated plant from the nature.

The extract can be extracted from various organs and extracted from, for example, roots, famines, stems, leaves, fruits and the like, and it is preferable that the extract is extracted from a leafy area of a mulberry leaf.

The extract of Sclerotinia spp. Contained in the composition of the present invention can be extracted by a known extraction method, and the method is not limited thereto. For example, the extract of the mulberry juice extract may be an extract obtained by pulverizing the leaves of the mulberry juice, which has been washed and dried to remove the foreign material, from water, a C _1-4 lower alcohol or a mixed solvent thereof. have. Specifically, water, methanol, ethanol, water and ethanol can be mixed daily. Although extracts using organic solvents contain a large amount of non-polar terpene compounds, extracts containing alcohol or a mixed solvent of water and ethanol contain a high content of phenolic compounds.

Further, in the present invention, the above-mentioned extract includes any one of the extract obtained by the extraction treatment, the diluted solution of the extract, the concentrate, the dried product obtained by drying the extract, or the adjusted product or the purified product thereof. The extraction method is not particularly limited, and extraction can be carried out at room temperature or with heating under conditions where the active ingredient is not destroyed or minimized. More specifically, the method for obtaining the Scutellaria baicalensis extract in the present invention is as follows. The extraction temperature is 20 to 100, specifically 30 to 80, and the extraction period is about 2 to 20 times the volume of water, lower alcohol, or a mixed solvent thereof, Extraction is performed using extraction methods such as shaking extraction, hot water extraction, cold extraction, reflux cooling extraction, or ultrasonic extraction for 10 to 10 days, preferably 4 to 8 hours.

The caffeoquinic acid compound of the present invention contains a large amount of phenolic compound caffeoylquinic acid, and the caffeoquinic acid compound is 3,4-di-O-dicaffeoylquinic acid (3, Di-O-dicaffeoylquinic acid, 3,5-di-O-dicaffeoylquinic acid, 4,5-di-O-dicaffeoylquinic acid ( 5-O-caffeoyl quinic acid, 3-O-caprooyl quinic acid, and 3-Op-coumaro quinic acid. And 3-Op-coumaroylquinic acid. In addition, the extracts of astringent juice contain astragalin and kaempferol which are flavonoid compounds.

The fractions of the present invention can be obtained by fractionating the fractions from the fractions of the fragments of the fragments. More specifically, the fraction fractions obtained by fractionating the fragments of the fragments with the use of a solvent such as ethyl acetate, hexane, chloroform or butanol have. The fraction may be obtained by a method known in the art, for example, after suspending a methanol extract of Asteraceae in distilled water, adding thereto about 1 to 100 times, specifically about 1 to 5 times the volume of the suspension, of hexane, ethyl Acetone, chloroform, butanol or the like is added thereto, and the solvent-soluble layer is extracted and separated one to ten times, more specifically two to five times. Further, a normal fractionation process may be further performed.

In one embodiment of the present invention, the fraction may be a fraction obtained by successively fractionating a Scutellaria burcin extract with at least two solvents selected from the group consisting of hexane, chloroform, ethyl acetate, butanol and water.

In one embodiment of the present invention, the fraction may be a butanol fraction obtained by fractionating the Asteraceae extract with water and chloroform, and then sequentially fractionating the water layer with butanol.

Thereafter, the above-mentioned Scutellaria baicalensis fraction can be separated and purified by silica gel chromatography to isolate the compound. When the silica gel chromatography for the compound separation is carried out, a mixed solvent of chloroform, methanol and water may be used as the mobile phase, and a mixed solvent of methanol and water may be used in the additional chromatography. The chromatography can be carried out once to several times until a single compound is purified, and concentration and recrystallization can be carried out if necessary.

The isolated compound is a flavonoid compound, and specifically, kaempferol 3-O-L-rhamnopyranoisde, Kempherol 3-O-D-galactopyridine O-D-glucopyranoisde, kaempferol 3-O-Dgalactopyranoside, and kaempferol 3-O-D-glucopyranoisde.

The metabolic disease may be a lipid-related metabolic disease, for example, fatty liver due to high fat, type 2 diabetes, hyperlipidemia, cardiovascular disease or atherosclerosis.

The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. The composition comprising a pharmaceutically acceptable carrier can be of various oral or parenteral formulations. 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, . In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical composition may be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories It can have one formulation.

The composition of the present invention is administered in a pharmaceutically effective amount.

The term "pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will vary depending on the species and severity, age, sex, , 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. However, for the desired effect, the extract of the present invention or its fractions may be administered at a dose of 1 to 200 mg / day, preferably 10 to 100 mg / day.

The composition of the present invention may be administered in the form of an individual therapeutic agent or in combination with other therapeutic agents exhibiting an effect of preventing or treating obesity or metabolic diseases, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. 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 adverse effect, and can be easily determined by those skilled in the art.

The term "individual" as used herein means all animals, including humans, who have already developed or are capable of developing a disease that can be prevented or treated through an anti-obesity or anti-metabolic disease activity and includes an extract or fraction of the present invention By administering the composition to a subject, the disease can be effectively prevented and treated.

The route of administration of the composition may be administered via any conventional route so long as it can reach the target tissue. The composition of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonarily, or rectally, though it is not intended to be limited thereto. The composition may also be administered by any device capable of transferring the active agent to the target cell.

The health functional food of the present invention includes an extract of Myringae sp., And a suitable food supplementary additive may be included.

The term "food supplementary additive " in the present invention means a component which can be added to foods in a supplementary manner, and it can be appropriately selected and used by those skilled in the art as added to produce health functional foods of each formulation. Examples of food aid additives include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, and carbonating agents used in carbonated beverages. However, the types of the food-aid additives of the present invention are not limited by these examples.

A health functional food may be included in the food composition of the present invention. The term "health functional food" in the present invention refers to a food prepared and processed in the form of tablets, capsules, powders, granules, liquids and circles by using raw materials and components having useful functions in the human body. Here, the term "functionality" means that the structure and function of the human body are controlled to obtain nutritional effects or effects useful for health use such as physiological actions. The health functional food of the present invention can be prepared by a method commonly used in the art and can be prepared by adding raw materials and ingredients that are conventionally added in the art. In addition, unlike general medicines, there is an advantage that there is no side effect that may occur when a medicine is taken for a long time by using food as a raw material, and it is excellent in portability. Therefore, the health functional food of the present invention is useful for improving anti- It can be taken as an adjuvant.

The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). In general, when preparing a foodstuff, the extract or its fraction according to the present invention is added in an amount of 1 to 10% by weight, preferably 5 to 10% by weight, of the raw material composition. However, in the case of long-term ingestion intended for health and hygiene purposes or for the purpose of controlling health, the amount can also be used in the above-mentioned range.

There is no particular limitation on the kind of the food. Examples of the food to which the above substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.

The health food composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient as well as ordinary foods. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 of the composition of the present invention.

In another embodiment of the present invention, the present invention provides a use for the prophylaxis or treatment of obesity or metabolic diseases of the Fusarium spp. Extract or its fractions. The extracts or fractions thereof according to the present invention reduce body weight, reduce body fat, reduce plasma glucose and total cholesterol levels, reduce fat accumulation in the body (liver and visceral fat tissue) And by reducing gene expression involved in adipocyte formation, it can be very useful for prevention or treatment of obesity or metabolic diseases.

Hereinafter, the present invention will be described in more detail with reference to Examples of the present invention and Comparative Examples which are not based on the present invention, but the scope of the present invention is not limited by the following Examples.

Example 1: Preparation of an extract of a scabbard

The leaves (500 g) of Aster glehni ( Ulleungdo) were dried and then ground and extracted with methanol (MeOH) for 60 hours under reflux. The extract was filtered, concentrated under reduced pressure and lyophilized to obtain a solid methanol extract (73 g). 800 mL of water and chloroform (CHCl ₃ ) were added to the methanol extract, and the mixture was separated into a water layer and a chloroform layer. 800 mL of butanol was added to the double water fraction layer to obtain a butanol (BuOH) layer (10.5 g).

<Experimental Method>

(1) Cell culture

3T3-L1 mouse preadipocytes were cultured in DMEM supplemented with 10% CO (100 ng / ml) using 10% bovine calf serum, 100 g / ml streptomycin and 100 units / ml penicillin added Dulbeccos Modified Eagles medium ₂ &lt; / RTI &gt; Cells were confluently grown in 10% bovine serum / DMEM. Two days after confluence (Day 0), cells were resuspended in MDI induction medium (10% fetal bovine serum, 0.5 mM 2-isobutyl-1-methylxanthine (IBMX), 1 μM dexamethasone and 1 μg / DMEM). Two days after MDI (Day 2), the medium was replaced with insulin medium (DMEM containing 10% fetal bovine serum and 1 ug / mL insulin). Two days later (Day 4), the medium was replaced with 10% FBS / DMEM. Cells were fed with 10% FBS / DMEM every 2 days. Full differentiation was completed on Day 8.

(2) Oil-red O. Dyeing

The cells were stained with Oil Red O to look for adipocyte-forming ability and fat-accumulating ability. On Day 8, the cultured cells were washed three times with phosphate buffered saline (PBS) and fixed with 4% formaldehyde for 30 minutes at room temperature. Cells were washed three times with PBS and stained with 0.5 ug / mL Oil Red for 15 minutes. After washing with PBS, representative micrographs were captured using a microscope and the degree of fat accumulation was measured.

(3) AdipoRed assay method

3T3-L1 cells were plated at a density of ³ × 10 ⁵ cells per well on a 6-well plate and the effect on the differentiation of the cells in the presence and absence of the Aspergillus sieboldii extract was examined. At differentiation Day 2 and Day 8, cells were treated with AdipoRed (Lonza, Houston, TX, USA) and analyzed for intracellular triglycerides (TG).

(4) Real-time PCR analysis

Total RNA was isolated from differentiated 3T3-L1 cells and epididymal adipose tissue using Easy-Blue reagent (Intronbiotechnology Inc., Kyonggi-do, Korea).

Total RNA was amplified using Epoch And quantified using a micro-volume spectrophotometer system (BioTek Instruments, Inc., Winooski, VT, USA). Equal amounts of total RNA were converted into cDNA using a high performance cDNA reverse kit (Applied Biosystems, Foster City, Calif., USA) (incubation for 25 min at 10 min, incubation at 50 캜 for 90 min and 85 캜 for 5 min Condition). Real-time PCR was performed with a Step One Plus real-time PCR system (Applied Biosystems, Foster City, CA, USA). SYBR Green Mastermix (Bio-Rad, Hercules, CA, USA) and primers (Santa Cruz, CA, USA) were used for PCR analysis (Santa Cruz, CA, USA). The reactions proceeded under the following conditions, respectively:

Initial denaturation was performed at 95 ° C for 10 minutes, at 95 ° C for 5 seconds, at 40 cycles, at 60 ° C for 45 seconds; And the final melting curve is 15 seconds at 95 ° C, 1 minute at 60 ° C, 15 seconds at 95 ° C for cells, 30 cycles at 94 ° C for 30 seconds, 30 seconds at 60 ° C, 30 seconds at 72 ° C Seconds) and initial denaturation for 5 minutes.

Changes in gene expression were measured using a comparative threshold cycle (Ct) method (Applied Biosystems), and each measurement was normalized to the value of GAPDH.

(5) Western blot analysis

3T3-L1 cells were lysed with lysis buffer (25 mM HEPES; pH 7.4, 100 mM NaCl, 1 mM EDTA, 5 mM MgCl ₂ , 0.1 mM dithiothreitol and protease inhibitor mixture). The quantificated protein was immersed in a Tris-glycyl SDS-polyacrylamide gel and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membranes were then blocked for 1 hour at room temperature with 5% skim milk in Tris-buffered physiological saline containing 0.1% Tween 20 (TBST) and incubated overnight at 4 ° C with 1: 1000 dilution primary antibody . The next membrane was washed three times for 10 minutes each with TTBS and reacted for 2 hours with 1: 2500 dilution of the secondary antibody. After the reaction, complexes were visualized using ECL plus detection reagents (GenDEPOT, Barker TX, USA).

(6) Animal experiment

3-5 weeks old male C57BL / 6J mice of 15-17 g were purchased from BioLink (Daejeon, Korea) and used. All animals were subject to the NIH guidelines and guidelines for laboratory animal use and management. Mice (5 cages) were maintained in a constant temperature condition of 23 ° C ± 2 ° C for 12 hours / cancer cycle, and the humidity was 55 ± 10%. The mouse allowed free access to food and tap water. After 1 week acclimation period, 40 mice were randomly divided into 4 groups as shown in the following table, and 10 mice were divided into 1 group, and they were bred for 10 weeks.

Weekly weight and dietary intake were recorded. The animals were fasted overnight on the expiration date of the experiment. The next day, mice were anesthetized and blood samples were collected with cardiac puncture. Blood samples were centrifuged at 3000 rpm for 10 minutes to separate the plasma. Glucose and total cholesterol analysis was performed in serum samples according to an enzymatic method using a commercial kit (Biovision Research Products, Inc, CA, USA). Mice were sacrificed by cervical rupture, liver, epididymal, total fat pads were excised and weighed and immediately stored at 80.

(7) Histological analysis

Epididymal fat and liver tissue were fixed with 4% paraformaldehyde and impregnated with paraffin. The fat cells were cut into 4m and 8m sections for analysis of diameter, surface area and number, and stained with hematoxylin and eosin (H & E). The stained slides were observed and photographed on an SZX10 microscope (Olympus, Seoul, Korea).

(8) Statistical analysis

Experimental results were expressed as mean ± SEM. The results were analyzed by one-way ANOVA. Statistical analysis software (Version 19.0) was used for statistical analysis. p <0.05 was considered statistically significant.

Experimental Example 1. Effect on adipocyte production

Adipocyte differentiation was carried out using 3T3-L1 cells in order to see if the extract of Aspergillus sieboldii had an anti-obesity effect. When 3X10 ⁵ cells were divided into 6 wells and confluent, differentiation was induced as described in the experimental method. The differentiation of preadipocytes was induced in the presence or absence of the extracts of. After 8 days, 0.5 μg / mL Oil Red (Sigma aldrich) was added to the cultured cells and fat accumulation and intracellular triglycerides (TG) were analyzed. Troglitazone, anti-diabetic and anti-infectious agents (van Tits et al., 2005) were used as positive control for fat accumulation inhibition.

Experimental results showed that the number and size of lipid droplets of cytosol were significantly reduced (see Figure 1A). And fat accumulation also decreased in a concentration-dependent manner (see FIG. 1B). Intracellular TG was analyzed by adipo-red assay method. The degree of accumulation of TG in 2-day differentiated cells treated with Mugwort extract was not different from that of group 2 (see FIG. 1C). However, in the cells differentiated for 8-day, TG was down-regulated markedly in a concentration-dependent manner (see FIG. 1D).

Experimental Example 2 Effect of adipocyte on expression of adipocyte-forming gene

The differentiation of adipocytes into adipocytes involves the expression of adipocyte-forming genes. First, PPAR, C / EBP and SREBP1c are key regulators in adipocyte formation, and LXR is also involved in adipocyte formation. The expression levels of adipocyte markers were measured by Real-time PCR and Western blotting after differentiation for 8 days in the presence or absence of 100, 200, and 400 μg / After the cells were treated for 8 days with an extract of Mortalia japonica, the degree of mRNA expression of PPAR, C / EBP, SREBP1c and LXR was examined. As a result, mRNA expression of the gene was decreased (See FIG. 2A). Through the above experiment, it can be seen that the expression of the adipocyte-forming (related) gene is regulated in the adipocyte in the case of the group treated with the extract of Aspergillus oryzae.

Experimental Example 3. Effect of weight, visceral fat pad and liver weight reduction

Figure 3 shows the weight, the increase in body weight, and the degree of change in visceral fat pad and liver weight. In the HFD group, body weight, fat weight and liver weight were significantly increased compared to the control group. In group 3 (AG1 group), the weight gain was lower than the weight gain of the HFD group (see Figures 3A and 3B). The visceral weight and liver weight were lower in group 3 (AG1 group) compared to the HFD group (see Figs. 3C and 3D). The weight gain of group 4 (AG5 group) was significantly lower than that of HFD and control group (see FIGS. 3A and 3B).

The above experiment shows that the body weight, visceral fat pad, and liver weight of HFD-induced obesity mice are reduced in the case of the extract of the Aspergillus oryzae.

Experimental Example 4. Effect on fat accumulation

Representative liver and adipose tissues were stained with H & E to determine whether the decrease in body weight, visceral weight, and liver weight was due to a decrease in fat accumulation. As shown in FIG. 4, unlike the control group, the representative liver section of the HFD group clearly showed accumulation of fat particularly in hepatocytes around the central vein. Considering cytoplasmic hepatocyte vacuole, histological liver images of Group 3 (AG1 group) and Group 4 (AG4 group) showed that the Sclerophyllum sp. Extract alleviated fat accumulation (FIG. 4A). In fat tissues, the effect of the extract of Aspergillus oryzae on lipolysis was easy to see. The diameter and number of fat droplets in the adipose tissue of group 3 (AG1 group) and group 4 (AG4 group) were significantly reduced compared to the adipose tissue of the HFD group (see FIG. 4B). HFD-induced fat accumulation in the liver and adipose tissue was alleviated in the case of the extract of the mulberry juice extract.

EXPERIMENTAL EXAMPLE 5. Effect of Reducing Glucose and Total Cholesterol Level in Hepatic Plasma

Plasma glucose and total cholesterol levels were increased in the HFD group. In group 4 (AG5 group), the increase in glucose levels was significantly lower than in the HFD group. In group 3 (AG1 group), glucose levels were lower than the HFD group, but not statistically significant (see FIG. 5A). The cholesterol level of the island mugwort extract treatment group was lower than that of the HFD group, but not statistically significant (see FIG. 5B).

Experimental Example 6. Regulation of signal transducer involved in adipocyte formation

Real-time PCR analysis was performed to investigate the effect of the extracts of Aspergillus oryzae on HFD-induced adipocyte formation mRNA expression in epididymal white adipose tissue. In addition to its effect on adipocyte formation in vitro, it has been shown that mulberry juice extract weakens mRNA expression associated with adipocyte formation. The expression of PPAR was markedly decreased in the epididymal tissues removed from the sclerotin extract-treated HFD mice. Expression of C / EBP, a downstream target of PPAR, also remarkably decreased. Other adipocyte formation markers such as SREBP1c, LXR, LPL, and leptin were significantly reduced in the epididymal tissues of mice treated with Asteraceae extract (see Fig. 6A). In parallel with the PCR results, the expression of PPAR and C / EBP decreased in the epididymal adipose tissues from HFD mice treated with Asteraceae extracts compared to the HFD group.

Production Example 1. Preparation of powder

Island mugwort extract 10mg

Sucrose 100 mg

Talc 10mg

The above components are powdered and mixed, and filled in an airtight container to prepare a powder.

Production Example 2. Preparation of tablets

Island mugwort extract 10mg

Starch 100mg

Sucrose 100 mg

Magnesium stearate 2 mg

The tablets are prepared by mixing the above components according to a conventional method for producing tablets and then tableting them.

Preparation Example 3. Preparation of capsules

Island mugwort extract 10mg

Crystalline cellulose 3 mg

Lactose 15mg

1 mg of magnesium stearate

The above components are mixed according to a conventional method for preparing a capsule, and then filled in a gelatin capsule to prepare a capsule.

Production Example 4. Preparation of Granules

Island mugwort extract 10mg

Soybean extract 50mg

200 mg of glucose

Starch 500 mg

After mixing the above components, 100 ml of 30% ethanol is added and the mixture is dried at 60 to form granules, and the granules are filled by filling the granules.

Production Example 5. Preparation of a pellet

Island mugwort extract 20mg

Lactose 1,500mg

1,500 mg of glycerin

Starch 980 mg

After mixing the above components, the mixture is prepared to be 4 g per one ring according to a conventional method for producing a pellet.

Production Example 6. Preparation of injection

Island mugwort extract 10mg

180 mg mannitol

Sterile sterilized water for injection 2,970mg

Na ₂ HPO _{4 12} H ₂ O 30 mg

The above components are mixed and prepared so as to be 2 mL per ampoule according to a usual injection preparation method.

Production Example 7. Production of liquid agent

Island mugwort extract 10mg

10,000 mg per isomerization

Mannitol 5,000 mg

Purified water quantity

Dissolving the above components in purified water according to a usual liquid preparation method, adding an appropriate fragrance, filling the bottle and sterilizing it.

Claims (16)

Aster glehni ) extract or a fraction thereof as an active ingredient for the prevention or treatment of obesity or metabolic diseases.
The method according to claim 1,
A pharmaceutical composition for preventing or treating obesity or a metabolic disease, wherein the extract is a leaf extract of a scabbard.
The method according to claim 1,
Wherein the extract is extracted with water, a C _{1 -4} lower alcohol or a mixed solvent thereof.
The method according to claim 1,
Wherein the extract is extracted with at least one solvent selected from the group consisting of water, methanol, ethanol, and a mixed solvent thereof, and a pharmaceutical composition for preventing or treating obesity or metabolic diseases.
The method according to claim 1,
Wherein said fraction is a fraction obtained by successively fractionating a pine needle extract with at least two solvents selected from the group consisting of hexane, chloroform, ethyl acetate, butanol, and water, and a pharmaceutical composition for preventing or treating obesity or metabolic diseases Composition.
The method according to claim 1,
Wherein said fraction is a butanol fraction obtained by fractionating a mulberry juice extract with water and chloroform, and then fractionating the water layer with butanol in order to prevent or treat obesity or metabolic diseases.
The method according to claim 1,
The pharmaceutical composition for preventing or treating obesity or metabolic diseases, wherein the metabolic disease is a lipid-related metabolic disease.
The method according to claim 1,
Wherein the metabolic disease is at least one disease selected from the group consisting of fatty liver caused by high fat, type 2 diabetes, hyperlipidemia, cardiovascular disease, and arteriosclerosis, or a pharmaceutical composition for preventing or treating obesity or metabolic diseases.
Aster glehni ) extract or a fraction thereof as an active ingredient for preventing or ameliorating an obesity or metabolic disease.
10. The method of claim 9,
Wherein the extract is a leaf extract of a scabbard, and the health functional food for preventing or ameliorating an obesity or metabolic disease.
10. The method of claim 9,
Wherein the extract is extracted with water, a C _{1 -4} lower alcohol or a mixed solvent thereof.
10. The method of claim 9,
Wherein the extract is extracted with at least one solvent selected from the group consisting of water, methanol, ethanol and a mixed solvent thereof.
10. The method of claim 9,
Wherein the fraction is a fraction obtained by successively fractionating a pine needle extract with at least two kinds of solvents selected from the group consisting of hexane, chloroform, ethyl acetate, butanol and water, and a health function for preventing or improving obesity or metabolic diseases food.
10. The method of claim 9,
Wherein said fraction is a butanol fraction obtained by fractionating a pine needle extract with water and chloroform, and then fractionating the water layer with butanol in order to prevent or ameliorate an obesity or metabolic disease.
10. The method of claim 9,
Wherein the metabolic disease is a lipid-related metabolic disease, and a health functional food for preventing or ameliorating an obesity or metabolic disease.
10. The method of claim 9,
Wherein the metabolic disease is at least one disease selected from the group consisting of fatty liver caused by high fat, type 2 diabetes, hyperlipidemia, cardiovascular disease, and arteriosclerosis, or a health functional food for preventing or ameliorating an obesity or metabolic disease.

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