KR20170020995A - Composition for anti-obesity comprising extract of Hoveniae Semen cum Fructus as an effective component - Google Patents
Composition for anti-obesity comprising extract of Hoveniae Semen cum Fructus as an effective component Download PDFInfo
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- KR20170020995A KR20170020995A KR1020150115212A KR20150115212A KR20170020995A KR 20170020995 A KR20170020995 A KR 20170020995A KR 1020150115212 A KR1020150115212 A KR 1020150115212A KR 20150115212 A KR20150115212 A KR 20150115212A KR 20170020995 A KR20170020995 A KR 20170020995A
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- extract
- obesity
- methylene chloride
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
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Abstract
The present invention relates to an anti-obesity composition comprising an effective amount of a platelet extract as an active ingredient, and more particularly, to a pharmaceutical composition for anti-obesity or a health food containing an extract of methylene chloride at the top as an active ingredient. Since the gastrointestinal methylene chloride extract according to the present invention has an effect of inhibiting the differentiation of adipocytes derived from fibroblasts and inhibiting the expression of an adipocyte differentiation regulator gene or protein, the functional food for preventing and inhibiting obesity, The present invention can be used as a pharmaceutical composition for the treatment of adult diseases.
Description
The present invention relates to an anti-obesity composition comprising an effective amount of a platelet extract as an active ingredient, and more particularly, to a pharmaceutical composition for anti-obesity or a health food containing an extract of methylene chloride at the top as an active ingredient.
Obesity is a disease caused by the imbalance of food intake and energy use, and can be accompanied by serious illnesses such as cancer, type 2 diabetes and arteriosclerosis (Kopelman, 2000). Recently, obesity, diabetes, dyslipidemia, and hypertension have been called metabolic syndrome. Despite the worldwide efforts to understand and respond to the causes of obesity, obesity is accelerating. The WTO warns that "Obesity is a disease that requires long-term transparency." Obesity is not simply a cosmetic issue, but a cause of various diseases, increasing the perception that it is a serious life-threatening disease. The World Health Organization (WHO) says 17 million people die each year from cardiovascular disease, an important risk factor for obesity, and warns that in 2015, 1.5 billion people worldwide, up by 50 percent, could suffer from obesity.
Obesity has been reported to be caused by complex factors such as genetic, social, and economic factors, as well as social diminution of exercise and failure to control energy homeostasis due to increased intake of high calorie food. Recently, the worldwide incidence of obesity associated with metabolic syndrome including type 2 diabetes, which is known to be caused by ingestion of high-calorie foods and deterioration of physical activity, is increasing. In 2025, the population with these metabolic syndrome doubles to about 300 million. Obesity is a result of the unbalance of energy intake and consumption of the human body, resulting in an increase in fat cell size due to fat accumulation and an increase in the number of adipocytes due to the differentiation of fibroblasts into adipocytes. The adipogenesis process of fibroblasts differentiates into adipogenesis, which involves liver X receptor α (LXRα), CCAAT enhancer-binding protein-α (C / EBPα), peroxisomeproliferators-activatedreceptor- γ (PPARγ) (Srebp-1c) are involved (Ntambi and and Kim, 2000; Rosen et al., 2000; Seo et al., 2004). These transcription factors are mostly expressed in adipose tissue and their activation when differentiating into adipocytes is the same as fatty acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD-1), and acetyl-CoA carboxylase- (Schultz et al., 2000), which directly enhances expression of synthetic genes and increases fat accumulation in differentiated adipocytes (Joseph et al., 2002). For this reason, in order to develop an anti-obesity agent, many studies have been conducted to find natural substances that inhibit the activity of transcription factors or genes involved in adipocyte differentiation or lipogenesis (Gonzalez-Espinosa de los Monteros et al. 2011; Niwano et al., 2009).
Currently, various methods for treating obesity have been attempted. In general, the treatment of obesity includes diet therapy, exercise therapy, appetite suppressant, diuretic, diarrhea, or drug therapy using fiber to give a feeling of fullness, behavior modification therapy to correct wrong eating habits and lifestyle, Surgery for reducing the volume of fat, and fat removal surgery such as decomposing and removing fat cells using ultrasound in plastic surgery. In addition, amphetamine, reductil, thiazolidinedione, metformin, and the like, which are associated with obesity treatment, have shown various side effects. For this reason, interest in alternative approaches including the use of medicinal plants in relation to the treatment of obesity has been increasing recently, It is necessary to develop a new preventive and remedy for obesity from natural products that have been approved for stability.
The object of the present invention is (a) a method of extracting Hoveniae Semen cum Fructus by hot water extraction followed by lyophilization; (b) suspending methanol in the geothermal water extract prepared in the step (a) to obtain an endogenous methanol extract; And (c) sequentially fractionating the mitochondrial methanol extract with n-hexane and methylene chloride.
Another object of the present invention is to provide a pharmaceutical composition for preventing or treating obesity, which comprises the gefitogen methylene chloride extract prepared by the above method as an active ingredient.
Further, the object of the present invention is to provide a functional health food for preventing or ameliorating obesity, which comprises the geomagnetic methylene chloride extract prepared by the above method as an active ingredient.
In order to accomplish the object of the present invention, the present invention provides a method for preparing a microcrystalline cellulose, comprising the steps of: (a) hot-water extraction and freeze-drying Hoveniae Semen cum Fructus; (b) suspending methanol in the geothermal water extract prepared in the step (a) to obtain an endogenous methanol extract; And (c) sequentially fractionating the topoisomerase methanol extract with n-hexane and methylene chloride.
In one embodiment of the present invention, the hot water extraction in the step (a) may be performed at a temperature of 90 to 100 ° C. for 7 to 9 hours twice.
The present invention provides a pharmaceutical composition for preventing or treating obesity, which comprises the geomagnetic methylene chloride extract prepared by the above method as an active ingredient.
In one embodiment of the present invention, the geomagnetic methylene chloride extract may be contained at a concentration of 0.1 to 10 μg / ml.
In one embodiment of the present invention, the gastrointestinal methylene chloride extract may have the ability to inhibit PPARγ, C / EBPα, aP2, adiponectin and resistin, the adipocyte differentiation regulatory genes.
The present invention provides a functional health food for preventing or ameliorating obesity, which comprises the geomagnetic methylene chloride extract prepared by the above method as an active ingredient.
The present invention relates to a method for preventing or inhibiting obesity, comprising administering to a patient in need thereof an effective amount of a functional food for preventing or inhibiting obesity, , Or as a pharmaceutical composition for the treatment of obesity and adult diseases caused thereby.
FIG. 1 shows the cytotoxicity of 3T3-L1 cells to the fractions isolated from the extracts of endosperm.
FIG. 2 shows the results of measuring the amount of fat accumulated in the cells after 3T3-L1 cells were treated with the fractions separated from the endoplasmic reticulum extract.
FIG. 3 shows the results of confirming the degree of accumulation of intracellular fat at various concentrations of the methylene chloride fraction extract and the n-hexane fraction fraction among the fractions separated from the endogenous extract.
FIG. 4 shows the results of examining the gene expression levels of PPARγ and C / EBPα, which are regulators of adipocyte differentiation, by treating a 3T3-L1 adipocyte with methylene chloride extract.
FIG. 5 shows the results of analysis of the expression levels of transcription factors involved in obesity by RT-PCR.
FIG. 6 shows the results of Western blot analysis of the expression levels of PPARγ and C / EBPα, which are regulators of adipocyte differentiation, by treating a 3T3-L1 adipocyte with methylene chloride extracts.
FIG. 7 shows the results of Western blot analysis of protein expression levels of aP2, adiponectin and resistin, which are regulators of adipocyte differentiation, by treating mitochondrial chloride extracts of 3T3-L1 adipocytes.
The present invention relates to a novel use of a Zygomycota extract or a Zygomycota extract which has been found to have excellent anti-obesity activity.
In the present invention, attention has been paid to a diabetic extract as a new therapeutic agent for obesity or prophylactic agents. Globules are often referred to as Hoveniae semen cum fructus, and are frequently used as herbal medicines in oriental medicine.
The earth is tasted or tasted, and the nature is plain, and it mainly goes into the heart, the rain, and the menopause to exert its medicinal effect, to quench the thirst, to eliminate the frustration, to cleanse the heat, to release the digestion and to pass through the stool and urine . The first reference to the district party has referred to "prime Darling 新修本草" in contemporary 唐代, "global aesthetic pyeongmu solo dupung sobok mulgeup 枳味甘平無毒主頭風小腹物急". In addition, in the Tang Dynasty of the Tang Dynasty, "the main herb of food and drink ," also describes the adverse side effects of diabetes , which is recorded as " Daci foot rotten food ". In addition, the earth and temper who 性味 the sense 甘 or subtracting flat 甘酸平, gwigyeong 歸經 mainly core parenteral 心脾經, 入 mouth menopause 肺經 and 26), saving trouble jigal 止渴除煩, let cheongyeol 淸熱, poison解酒毒, Lee feces 利大小便 will be 治療 treating brass 醉酒, fever sogal 熱病消渴, alternately 煩渴, vomiting feces disadvantage 嘔吐大小便不利.
Recent studies on diabetes mellitus have shown that it is effective in protecting hepatic function such as jaundice, fatty liver, liver sclerosis, gastrointestinal disease and colitis, which is a side effect of excessive drinking, and is effective in alcohol decomposition and liver function recovery. And antimicrobial activity. However, there is no information about the effect of the anti-obesity on the earth.
The inventors of the present invention have confirmed that the extract of the present invention has anti-obesity activity. According to one embodiment of the present invention, 3T3-L1 adipocytes were treated with each fraction to measure fat accumulation inhibition, Methylene chloride and n-hexane in the fraction were found to be effective at inhibiting fat accumulation at 5 μg / ml (see FIG. 2). Based on the above results, the inhibitory effect of fat accumulation at various concentrations of methylene chloride and n-hexane fractions in the mitochondrial fractions was confirmed by Oil red O staining. As a result, methylene chloride and n-hexane fraction were found to be 5 μg / ml (Fig. 3). In particular, it was confirmed that the inhibitory effect of the methylene chloride fraction of the topoisomerase was excellent. These anti-obesity effects were all found in the methylene chloride fraction extracts of the earth, but the highest activity was observed especially at a concentration of 5 μg / ml.
Therefore, a composition comprising the extract of Zygomycota, especially the methylene chloride extract of Zymogen as an active ingredient, can effectively prevent or treat obesity.
In the present invention, 'obesity' means that the amount of fat in the body is excessively increased. Obesity is caused by an imbalance in the synthesis and degradation of fat in adipocytes. Obesity, which is caused by excess fat accumulation, increases the risk of developing chronic degenerative diseases and increases the risk of coronary artery disease, hypertension, stroke, hyperlipemia and diabetes .
In the present invention, " prevention " means all actions that inhibit obesity or delay progression by administration of the composition of the present invention.
&Quot; Improvement " in the present invention means all the actions of improving or ameliorating symptoms of obesity by administration of the composition of the present invention.
In the present invention, " treatment " means, unless otherwise indicated, reversing, alleviating, inhibiting or preventing the disease or disorder to which the term applies, or one or more symptoms of the disease or disorder And the term " treatment " as used herein refers to an act of treating when " treating " is defined as described above.
The term " administration " as used herein is meant to provide any desired composition of the invention to a subject in any suitable manner.
The term " individual " as used herein means all animals such as a human, a monkey, a dog, a goat, a pig, or a mouse having a disease in which symptoms of obesity can be improved by administering the composition of the present invention.
The term " pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit or risk ratio applicable to medical treatment, including the type of disease, severity, activity of the drug, The time of administration, the route and rate of excretion of the drug, the duration of the treatment, factors including drugs used simultaneously and other factors well known in the medical arts.
The extract of Zygomycota according to the present invention can be obtained by extracting and isolating from nature using an extraction and separation method known in the art, and the 'extract' defined in the present invention can be prepared by using an appropriate solvent, , And may include, for example, allergen extracts, polar solvent-soluble extracts, or non-polar solvent-soluble extracts.
Suitable solvents for extracting the extract from the kidney include, but are not limited to, water or an organic solvent. For example, purified water, methanol, ethanol, propanol, isopropanol isopropyl alcohol, isopropanol, butanol and the like, an alcohol having 1 to 4 carbon atoms, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride ), Hexane, and cyclohexane, may be used alone or in combination. However, most preferably, a methylene chloride solvent can be used.
As the extraction method, any one of the methods such as hot water extraction method, cold extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method and compression method can be selected and used. In addition, the desired extract may be further subjected to a conventional fractionation process or may be purified using a conventional purification method. There is no limitation on the method for producing the platelet extract of the present invention, and any known method can be used.
For example, the platelet extract contained in the composition of the present invention may be prepared in powder form by an additional process such as vacuum distillation, freeze-drying, or spray-drying, with the primary extract extracted by the hot water extraction or solvent extraction method described above have. Further, the primary extract can be further purified by using various chromatographies such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography and the like, You can get it.
Therefore, in the present invention, the termite extract is a concept including all the extracts, fractions and tablets obtained in each step of extraction, fractionation or purification, their diluted solutions, concentrates or dried products.
In the present invention, the Zygomycota extract or Zygote Methylene Chloride Extract may be prepared by any known method as described above, but most preferably (a) the Hoveniae Semen cum Fructus is extracted by hot water extraction Lyophilizing; (b) suspending methanol in the geothermal water extract prepared in the step (a) to obtain an endogenous methanol extract; And (c) sequentially fractionating the mitochondrial methanol extract with n-hexane and methylene chloride.
The composition of the present invention, which comprises the above-mentioned extract of Gejungjae or methylene chloride extract as an active ingredient, can be used as a pharmaceutical composition or a food composition.
The pharmaceutical composition of the present invention can be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned active ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, A lubricant or a flavoring agent can be used.
The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.
The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.
In one embodiment of the present invention, the platelet extract of the present invention may be contained in the composition at a concentration of 10 to 100 μg / ml, and the platelet extract of the present invention may be contained in an amount of 0.1 to 95% .
The obesity diseases in which the pharmaceutical composition of the present invention can exhibit therapeutic effects include obesity, diabetes, hyperlipidemia, fatty liver, arteriosclerosis, hypertension, cardiovascular disease, hyperinsulinemia, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia But are not limited to, a metabolic syndrome in which simultaneous occurrence of a disease, a metabolic syndrome, an endothelial dysfunction, a pre-aggregation state, dyslipidemia and atherosclerotic diseases.
The composition of the present invention may also be a food composition. In particular, it has been confirmed by one embodiment that the Zygomycota extract according to the present invention does not induce toxicity in cells and mice. Therefore, the food composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient, as well as conventional food compositions, in addition to containing an effective ingredient Jimae extract.
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. The above-described flavors can be advantageously used as natural flavorings (tau martin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.).
The food composition of the present invention can be formulated in the same manner as the above pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolates, foods, confectionery, pizza, ram noodles, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes, .
In addition, the above food composition may also contain flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and aging agents (such as cheese, chocolate, etc.) ), Pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks and the like. In addition, the food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.
The active ingredient of the present invention is a natural substance, which is a natural substance and has little side effects such as chemical agents. Therefore, it can be safely used for long-term use for prevention or treatment of obesity.
The present invention also provides a health functional food for the amelioration of an obesity disease, which comprises an extract of Zygomycota or a methylene chloride extract of Zymogen as an active ingredient.
The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and rings for the purpose of improving obesity diseases.
In the present invention, the term " health functional food " refers to foods manufactured and processed using raw materials or ingredients having useful functions in accordance with Law No. 6727 on Health Functional Foods. Or to obtain a beneficial effect in health use such as physiological action.
The health functional foods of the present invention may contain conventional food additives and, unless otherwise specified, whether or not they are suitable as food additives are classified according to the General Rules for Food Additives approved by the Food and Drug Administration, Standards and standards.
Examples of the items listed in the above-mentioned "food additives" include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as persimmon extract, licorice extract, crystalline cellulose, high color pigment and guar gum; L-glutamic acid sodium preparations, noodle-added alkalis, preservative preparations, tar coloring preparations and the like.
For example, the health functional food in the form of tablets may be prepared by granulating a mixture of the active ingredient of the present invention, Gejitae extract or Giemsa methylene chloride extract, with an excipient, a binder, a disintegrant and other additives in a conventional manner, A lubricant or the like may be put in a compression molding, or the mixture may be directly compression molded. In addition, the health functional food of the tablet form may contain a mating agent or the like if necessary.
The hard capsule of the capsule form of the health functional food can be prepared by filling a mixture of the active ingredients of the present invention, which is the active ingredient of the present invention, or the gentian methylene chloride extract with excipients such as excipients, A granule extract, or a mixture of a metazoan chloride extract with an additive such as an excipient may be filled in a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative and the like, if necessary.
The ring-shaped health functional food can be prepared by molding a mixture of the active ingredient of the present invention, Juza extract or Juzojie methylene chloride extract, excipient, binder, disintegrant and the like, according to a conventionally known method, It can then be coated with white sugar or other emulsions, or the surface can be coated with a material such as starch or talc.
The granular form of the health functional food may be prepared by granulating the granule extract of the present invention or the mixture of the extract of methylene chloride and the excipient, binder, disintegrant, etc. into granules by a known method. May contain flavoring agents, compatibilizing agents and the like.
As described above, the health functional food of the present invention, which contains the extract of the present invention or the methylene chloride extract of the present invention as an active ingredient, has no cytotoxicity and has the effect of inhibiting adipocyte differentiation and adipogenesis, It is effective for treatment.
The health functional food may be a beverage, a meat, a chocolate, a food, a confectionery, a pizza, a ramen, a noodle, a gum, a candy, an ice cream, an alcoholic beverage, a vitamin complex and a health supplement food.
The present invention also provides a method for the prevention or treatment of obesity diseases comprising administering to a mammal a Zymogen extract or a Zymogen methylene chloride extract.
The term "mammal " as used herein refers to a mammal that is the subject of treatment, observation or experimentation, preferably a human.
The term "therapeutically effective amount " as used herein refers to the amount of active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, physician or other clinician, The amount that induces the relief of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and the number of administrations of the active ingredient of the present invention will vary depending on the desired effect. Thus, the optimal dosage to be administered can be readily determined by those skilled in the art and will vary with the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, The age, body weight, sex, diet, time of administration, route of administration and fraction of the composition, duration of treatment, concurrent medication, and the like. In the treatment method of the present invention, in the case of an adult, it is preferable to administer the platelet extract of the present invention at a dose of 0.01 mg / kg to 250 mg / kg once or several times a day.
In the treatment method of the present invention, the composition comprising the extract of the present invention or the methylene chloride extract of the present invention as an active ingredient can be administered orally, rectally, intravenously, intraarterially, intraperitoneally, intramuscularly, intrasternally, transdermally, Lt; RTI ID = 0.0 > and / or < / RTI > intradermal route.
Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that the following examples are merely illustrative of the present invention and that the scope of the present invention is not limited to these examples.
< Example 1>
Preparation of experimental material
The water extracts of the globule used in the experiments of the present invention were supplied by Saarom Co. (Changheung, Jeonnam). The extraction conditions are as follows. After 1000 L of water was added to 100 kg of fresh water and heated at 95 ° C or higher for 8 hours, the water was removed and 500 L of water was added to the remaining aliquot, and the mixture was heated at 95 ° C or higher for 8 hours. Respectively. The yield of the geothermal water extract was 6%. For the fractionation, 30 g of water extract was suspended in 300 mL of 10% MeOH, and then 400 mL of 3-methylbenzene (400 mL × 3), ethyl acetate (400 mL × 3) and n-butanol mL x 3) were sequentially added to obtain an extract fraction. Each fraction was concentrated under reduced pressure and the final fractions were obtained using a Centrifugal Evaporator (EYELA, Japan) and dissolved in DMSO (dimethyl sulfoxide, (CH 3 ) 2 SO).
< Example 2>
Experimental Method
<2-1> Cytotoxicity test
For the measurement of cell viability, 3T3-L1 cells, preadipocyte, were cultured at 2 × 10 4 cells / well in a 96-well plate and cultured for 24 hours. methylene chloride, n-butanol, n-hexane) for 48 hours. 10 μl each of MTS (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphe-nyl) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt) And then cultured in a 5% CO 2 incubator at 37 ° C for 4 hours. After incubation, the absorbance was measured at 490 nm with an ELISA reader.
<2-2> Cell culture and differentiation
3T3-L1 cells were cultured in Dulbecco's modified Eagle's media (DMEM) containing 10% bovine serum (BS) and penicillin / streptomycin / glutamine (P / S / G) antibiotics Lt; 0 > C, 5% CO 2 incubator. The cells were washed with 1 × PBS at intervals of 2-3 days, and then 500 μl of 0.25% trypsin was treated to desorb and pass the cells.
(1 μg / ml), DEX (1 μM), and IBMX (0.5 mM) were added to the DMEM medium containing 10% fetal bovine serum (FBS) And then cultured for 2 days. After 2 days of culture, the medium was replaced with a medium containing only insulin, and the fraction extracted from the mitochondria was treated for 2 days. The medium was replaced with a medium containing only insulin, followed by culturing for 2 days. The medium was discarded and the remaining cells were recovered Was used for the experiment.
<2-3> Oil Red O staining
The fat in the cells differentiated into adipocytes was stained and the degree of differentiation was measured. Cells were fixed with 10% formaldehyde for 1 hour, washed with 60% isopropanol, completely dried and stained with 60% Oil Red O for 30 minutes. After 30 minutes, the cells were rinsed 3-4 times with tertiary distilled water. After completely dried, cells with red granules in the stained cytoplasm were regarded as differentiated adipocytes, and stained Oil Red O was dissolved in 100% isopropanol Then, the absorbance at 500 nm was measured to determine the degree of differentiation of adipocytes.
<2-4> RNA extraction and real-time RT- PCR
The adipocytes were differentiated, treated with different concentrations of the drug, and extracted with QIAzol lysis reagent. Single stranded cDNA was prepared using 2 μg of RNA obtained from each experimental group as a template and using a power cDNA synthesis kit (iNtRON Biotechnology, Seongnam, Kyunggi, Korea). The expression levels of mRNA were analyzed using real-time RT-PCR (Applied Biosystems, Foster City, CA, USA) with primers specific for PPARγ, C / EBPα, aP2, adiponectin and resistin. The primers used in the experiments are shown in Table 1.
<2-5> Protein extraction and Western Blotting
3T3-L1 cells were treated with radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris-HCl (pH 7.5), 0.1% sodium dodecyl sulphate (SDS), 0.1% Triton X- 1% Nonidet P-40, 0.5% sodium deoxycholate, 150 mM NaCl, 1 mM phenylmethysulphonyl fluoride]. The separated proteins (15 μg) were mixed with sample buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 20% glycerol, 10% 2-mercaptoethanol) and allowed to react at 95 ° C for 5 minutes. After electrophoresis on 10-12% SDS-polyacrylamide (PAGE) gels, they were transferred to polyvinyl difluoride (PVDF) membrane. After blocking with 5% skim milk, several primary antibodies (PPARγ, C / EBPα, aP2, adiponectin, and resistin) were attached and reacted overnight in a refrigerated chamber. The reaction was performed with a secondary antibody for 1 hour or longer, and the target protein was identified using ECL solution (Amersham Bioscience, Piscataway, NJ, USA).
<2-6> Statistical analysis
Experimental results were expressed as mean ± SEM for each experiment, and t- test was used for statistical analysis to determine the difference between the groups. The results of p <0.05 were considered statistically significant. All statistical analyzes were performed using
< Example 3>
Cytotoxicity test results
The MTS assay was performed to observe the effects of ethyl acetate, methylene chloride, n-butanol, and n-hexane fractions extracted from the mitochondria on fat cell survival.
As a result, it was confirmed that the extract fraction of ethyl acetate, methylene chloride, n-butanol and n-hexane was not cytotoxic up to 5 μg / ml and n-butanol was not cytotoxic up to 10 μg / ml. On the other hand, ethyl acetate, methylene chloride, and n-hexane extract fractions showed cytotoxicity at 10 μg / ml (see FIG. 1).
< Example 4>
Inhibitory effect of adipocyte differentiation
The present inventors treated 4T ethyl-acetate, methylene chloride, n-butanol, and n-hexane fractions extracted from mitochondria with 3T3-L1 adipocytes The degree of inhibition of adipocyte differentiation was measured. The degree of differentiation inhibition was confirmed by Oil Red O assay and the expression level of adipocyte differentiation factor was confirmed by real-time RT-PCR and Western blot.
<4-1> 3 T3 - L1 Inhibitory effect of fat precursor cell fat accumulation
Oil Red O staining method was used to measure the inhibition of fat accumulation in the mitochondrial fraction. 3T3-L1 adipocytes were treated with each fraction to determine the degree of fat accumulation inhibition. As a result, methylene chloride and n-hexane in the fraction showed inhibitory effect on fat accumulation at 5 μg / ml (See Fig. 2).
Based on the above results, the inhibitory effect of fat accumulation at various concentrations of methylene chloride and n-hexane fractions in the mitochondrial fraction was confirmed by Oil red O staining. As a result, methylene chloride and n-hexane fractions were found to be 5 μg / ml, and it was confirmed that the inhibitory effect of the methylene chloride fraction of the topoisomerase was excellent (see FIG. 3). Therefore, subsequent experiments with fat regulators were performed with methylene chloride extract from the Hoveniae Semen cum Fructus (MHF).
<4-2> MHF Adipocyte Differentiation regulator Gene Expression Inhibitory Effect
The adipogenesis process, which is differentiated from adipogenic precursor cells into adipocytes, is known to be induced by the gradual regulation of many adipogenic transcription factors. PPARγ and C / EBPα play the most important role in this differentiation process. These transcription factors are promoted by differentiation inducers such as hormones to differentiate adipose precursor cells into mature adipocytes. Expression of PPARγ and C / EBPα plays an important role in the expression of adipocytokines and adipogenic proteins such as adiponectin, aP2 and FAS, as well as morphological features such as intracellular lipid droplet production and cell size increase through interaction.
As a result of real-time RT-PCR, 3T3-L1 adipocytes were treated with MHF to measure the expression levels of PPARγ and C / EBPα, which are regulators of adipocyte differentiation. As a result, / EBP < / RTI > expression was suppressed. However, it was found that the expression of PPARy and C / EBPa could be most effectively inhibited at a concentration of 5 μg / ml (see FIG. 4).
In addition, it was also found that the expression of aP2, adiponectin, and resistin was most effectively inhibited by 5 μg / ml of MHF (see FIG. 5).
<4-3> MHF Adipocyte Differentiation regulator Inhibitory Effect on Protein Expression
The expression levels of PPARγ, C / EBPα, aP2, adiponectin, and resistin, which are regulators of adipocyte differentiation, were measured by Western blot analysis of MHF-treated 3T3-L1 adipocytes. C / EBPα, aP2, adiponectin, and resistin, and all of the factors were significantly inhibited from 1 μg / ml at 5 μg / ml of MHF (see FIGS. 6 and 7).
The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.
Claims (6)
(b) suspending methanol in the geothermal water extract prepared in the step (a) to obtain an endogenous methanol extract; And
(c) sequentially fractionating the mitochondrial methanol extract with n-hexane and methylene chloride.
Wherein the hot water extraction in step (a) is performed at a temperature of 90 to 100 캜 for 7 to 9 hours twice.
Wherein said guttae methylene chloride extract is contained at a concentration of 0.1 to 10 占 퐂 / ml.
Wherein said gastrointestinal methylene chloride extract has the ability to inhibit PPARγ, C / EBPα, aP2, adiponectin and resistin, the adipocyte differentiation regulatory genes.
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| KR101944295B1 (en) * | 2017-07-25 | 2019-01-31 | 계명대학교 산학협력단 | Composition for improving liver function comprising fermentation product of Hovenia dulcis fruit extract |
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| KR102556621B1 (en) | 2020-11-09 | 2023-07-17 | 경희대학교 산학협력단 | A composition for preventing or treating obesity comprising a mixed extract of Curcumae Radix and Syzygii Flos |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US919323A (en) | 1908-06-01 | 1909-04-27 | Frank B Cook | Tool for twisting wire-joints. |
| US1326271A (en) | 1919-12-30 | Implement-hitch |
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| US1326271A (en) | 1919-12-30 | Implement-hitch | ||
| US919323A (en) | 1908-06-01 | 1909-04-27 | Frank B Cook | Tool for twisting wire-joints. |
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| KR101944295B1 (en) * | 2017-07-25 | 2019-01-31 | 계명대학교 산학협력단 | Composition for improving liver function comprising fermentation product of Hovenia dulcis fruit extract |
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