KR101072604B1 - Compositions for the prevention and treatment of obesity comprising Widdrol - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of obesity, including a weed roll or a pharmaceutically acceptable salt thereof. Weedroll or a pharmaceutically acceptable salt thereof of the present invention is useful for the prevention and treatment of obesity because it has excellent lipolytic effect and inhibiting the differentiation of adipocytes into adipocytes.

Weedroll, obesity

Description

Compositions for the prevention and treatment of obesity comprising Widrol {Compositions for the prevention and treatment of obesity comprising Widdrol}

The present invention relates to a composition for the prevention and treatment of obesity, including a weed roll or a pharmaceutically acceptable salt thereof.

Diseases that account for the top group as the cause of death of modern people are reported to be caused by the imbalance of lifestyle, including diet. Nutrition consumed by proper diet is of utmost importance, and depending on the nutritional state, it is possible to reduce the morbidity rate and to heal the disease. Among them, obesity is usually defined as a BMI of 30 kg / m ² , a body fat amount of more than 25% of the body weight for men, 30% or more of the body weight for women. Obesity is a metabolic disease that is spreading rapidly in Korea and around the world due to changes in lifestyles. The World Health Organization (WHO) has already defined obesity as a disease in 1996. In 2015, it is estimated to reach 700 million.

The causes of obesity include genetic factors, eating habits, lifestyle, and imbalances in energy metabolism. The problems of obesity include diabetes, hypertension, hyperlipidemia, heart disease, arthritis, respiratory disease, sexual dysfunction, and cancer. It is recognized as a serious problem that not only threatens people's health as a risk factor to increase national health, but also affects national competitiveness by causing huge social expenses. Increasing social interest in the fight against obesity, and the large market potential of obesity treatments, domestic and overseas pharmaceutical companies are paying much attention to the development of obesity treatments. The anti-obesity drug is expected to form a market of about $ 2 billion worldwide and about 70 billion won in Korea in 2010, and the market size is expected to increase continuously. This trend is rapidly spreading in industrialized countries, where 5-10% of the total medical expenses are used in the obesity field, but there are no effective products on the market to meet the demand.

Currently, obesity treatments are divided into two markets: orlistat, which suppresses the digestion and absorption of fat, the cause of obesity, and sibutramine, which reduces food intake and increases energy consumption. As the development of engineering technology reveals a new mechanism for treating obesity, various active substances are being developed. Since most of the obesity drugs developed and marketed so far are chemically manufactured drugs, side effects and toxicity are problematic. Therefore, researches to develop obesity treatments from natural products are actively underway to solve these problems.

Accordingly, the inventors also found that the compound isolated from Juniperus chinensis was effective in anti-obesity while searching for an obese active substance from various medicinal plants and completed the present invention.

One object of the present invention relates to a pharmaceutical composition for the prevention and treatment of obesity, including weedroll or a pharmaceutically acceptable salt thereof.

Another object of the present invention relates to a method for preventing and treating obesity by administering the composition to a patient.

Another object of the present invention relates to a food composition for preventing obesity, including a weed roll or a pharmaceutically acceptable salt thereof.

In one embodiment, the present invention relates to a pharmaceutical composition for the prevention and treatment of obesity, including a widrol or a pharmaceutically acceptable salt thereof.

[Formula 1]

The "widdrol" of the present invention is a sesquiterpene compound as the compound of Formula 1. In order to identify the active ingredient exhibiting lipolytic activity in juniper extract, the present inventors fractionated a compound obtained by performing chromatography by adsorption chromatography on a non-polar solvent soluble extract of ultraviolet spectrophotometric spectrum, electron impact mass spectrometry, hydrogen nuclear magnetic resonance spectrum, carbon Analysis such as nuclear magnetic resonance spectrum was performed. Prior to the present invention, the present inventors have known the anticancer effect of withroll, but before the present invention has not been found in relation to the anti-obesity of withroll.

In the present invention, the term “pharmaceutically acceptable salts” means salts derived from pharmacologically or physiologically acceptable inorganic acids, organic acids and bases. Examples of suitable acid include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfuric acid Phonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Salts derived from suitable bases may include alkali metals such as sodium, alkaline earth metals such as magnesium, ammonium and the like.

Weedroll of the present invention can be separated from natural substances, preferably from plants. Various organs, roots, stems, leaves, flowers, as well as plant tissue cultures of natural, hybrid, and mutant plants can be extracted and isolated. In addition, chemical synthesis is possible by methods known in the art.

The method for obtaining a weed roll from a plant is by various known extraction methods. For example, the plant is ground and then extracted with a lower alcohol solvent such as methanol, ethanol and the like, which is then suspended in distilled water to give a nonpolar organic solvent such as normal hexane, ether, dichloromethane, chloroform, ethyl. It can be obtained by extracting and separating the nonpolar solvent soluble layer with an acetate or a mixed solvent thereof. From the obtained nonpolar organic solvent fraction, weedroll of formula (1) can be obtained. The extract can be further purified by performing one or more chromatographic chromatography, where the type of column and the developing solvent can be controlled in various ways (Harborne JB Phytochemical methods: A guide to modern techniques of plant analysis . 3rd Ed. pp 6-7, 1998).

In one specific embodiment, the present inventors finely chop the juniper and extract it with methanol, and then 1 to 10 times, preferably 2 to 5 times, with 1 to 100 times, preferably 1 to 5 times the volume of methylene chloride. Methylene chloride fractions obtained by ash extraction were separated by adsorption chromatography, such as silica gel column chromatography.

Withroll, a compound of the present invention, has a lipolytic effect. In a specific implementation, the lipolytic effect of weedroll was confirmed by decreasing triglyceride content and increasing glycerol amount in adipocytes. In addition, the expression level of lipolysis-related genes and fatty acid transfer genes were examined using microarrays. As a result, adenylate cyclase 8, phospholipase ₁ and phospholipase A, genes that induce lipolysis, were examined. ₂ , phospholipase C ₁ , lipoprotein lipase 1 expression was increased, and ACSL1 (acyl-CoA synthetase long chain family member 1), a fatty acid transport gene, was activated compared to the control group It was.

In addition, with a compound of the present invention, weedroll has an inhibitory effect on differentiation into adipocytes. In a specific implementation, it was confirmed that when the differentiation inducing agent and the weedroll were treated together in the step of differentiating 3T3-L1 adipocytes into complete adipocytes, the weedroll inhibited the adipocyte differentiation.

As described above, since the compound of the present invention, weedroll has a lipolytic effect and inhibits the differentiation into adipocytes, it is very effective in the prevention and treatment of obesity.

As used herein, the term “obesity” refers to a condition or disease with excess body fat due to energy imbalance. By administering the composition of the present invention, the breakdown of fat and differentiation into adipocytes are suppressed. You can lose weight with your back, and your blood cholesterol, triglycerides, low-density low-protein (LDL) -cholesterol in the blood is reduced.

As used herein, the term "prevention" means any action that inhibits or delays the development of obesity by administration of a composition. As used herein, the term "treatment" means any action that improves or advantageously changes the condition of obesity by administration of the composition.

The pharmaceutical composition for preventing and treating obesity of the present invention comprises 0.1 to 50% by weight of the compound based on the total weight of the composition. In addition, the composition does not increase the efficacy, but may include additional ingredients that are commonly used in the pharmaceutical composition to improve the smell, taste, time and the like. In addition, the composition is inorganic, organic such as vitamins B ₁ , B ₂ , B ₆ , C, E, niacin, carnitine, betaine, folate pantothenic acid, biotin, zinc, iron, calcium, chromium, magnesium, mixtures thereof Additives may further be included. In addition, the composition may include a substance having a therapeutic activity against diabetes alone or used previously.

The composition may comprise a pharmaceutically acceptable carrier and be formulated for human or veterinary use for oral or parenteral use.

When formulating the composition of the present invention, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may include at least one excipient such as starch, calcium carbonate ( Calcium carbonate, sucrose or lactose, and gelatin are mixed and prepared. In addition to simple excipients, lubricants such as magnesium, styrate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents, water and liquid paraffin. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and the like. As the non-aqueous solvent and suspending solvent, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, injectable esters such as ethyl oleate and the like can be used.

Such compositions may be presented in unit-dose (single) or multi-dose (several) containers, such as sealed ampoules and vials, and immediately before use, in sterile liquid carriers such as injectable water. Can be stored under freeze-drying conditions requiring only the addition of. Immediate injection solutions and suspensions can be prepared from sterile powders, granules and tablets.

As another aspect, the present invention relates to a method for preventing and treating obesity by administering to the patient the pharmaceutical composition comprising a weedroll of formula (1) or a pharmaceutically acceptable salt thereof.

As used herein, the term "patient" refers to humans and horses, sheep, pigs, goats, camels, who have diseases caused by obesity and their direct and indirect causes, and whose symptoms may be improved by administering the composition of the present invention. Means antelope, dog and other animals. By administering to a patient a composition comprising a weedroll of Formula 1 or a pharmaceutically acceptable salt thereof, the above-mentioned obesity can be effectively prevented and treated. In addition, the composition of the present invention can be administered in parallel with existing obesity treatments.

As used herein, the term "administration" means introducing a predetermined substance into a patient by any suitable method, and the route of administration of the composition of the present invention is oral or parenteral via any general route as long as the target tissue can be reached. May be administered. In addition, the composition may be administered by any device in which the active agent may migrate to the target cell.

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

As used herein, the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level refers to the sex, age, severity, activity of the drug of the patient. , Drug sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical arts. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. It may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art. The method of administering a composition comprising the compound of formula 1 or a pharmaceutically acceptable salt thereof of the present invention is preferably oral or intravenous administration. In general, the effective dose is once orally based on an adult. 1 to 500 mg / kg is preferable, and in the case of intravenous administration, 1 to 100 mg / kg is preferable. Dosage levels for a particular patient may vary depending on gender, age, health condition, diet, time of administration, method of administration, drug mixture and severity of disease.

As another aspect, the present invention relates to a food composition for preventing obesity of the above formula (Widrol) or a pharmaceutically acceptable salt thereof.

Weedroll of the formula (1) of the present invention or a pharmaceutically acceptable salt thereof has an excellent lipolytic effect as described above, and has the effect of inhibiting differentiation into adipocytes. Therefore, weedroll of Formula 1 or a pharmaceutically acceptable salt thereof may be added to food to prevent obesity.

When the food composition of the present invention is used as a food additive, the composition may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). Generally, in the preparation of food or beverages, the composition of the present invention is added in an amount of up to 15% by weight, preferably up to 10% by weight relative to the raw material. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, it may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

There is no particular limitation on the kind of food. Examples of the food to which the 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, drinks, Alcoholic beverages and vitamin complexes, and includes all health foods in the conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The natural carbohydrates described above may be used as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and natural sweeteners such as dextrin and cyclodextrin, and synthetic sweeteners such as saccharin and aspartame. . The proportion of the natural carbohydrate is generally about 0.01 to 0.4 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, And a carbonation agent used for the carbonated beverage. In addition, the composition of the present invention may contain a pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not critical but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Weedroll or a pharmaceutically acceptable salt thereof of the present invention is very useful for the prevention and / or treatment of obesity because it has a very excellent lipolytic effect and inhibiting the differentiation of adipocytes into adipocytes.

The invention will be explained in more detail through the following examples. These examples are presented by way of example only, so that the present invention may be more understood, these examples should not limit the scope of the invention.

Reference Example  : Cell Culture

Mouse embryo 3T3-L1 adipocytes used in the following runs were purchased from the American Type Culture Collection (ATCC), and DMEM (Dulbecco's modified) with 10% bovine calf serum (BCS) and 0.1% gentamycin added. Eagle's medium) was incubated at 37 ℃, 5% CO ₂ conditions.

Example 1 Isolation and Preparation of Widrol, a Compound of the Invention

Junirus chinensis ) was extracted with MeOH to separate the active ingredient with widrol, and concentrated under reduced pressure to obtain 713.6 g of the extract. By the addition of distilled water, the extract was then dissolved in CH ₂ Cl ₂ layer and the fraction H ₂ O and CH ₂ Cl ₂ layer 399.36 g, H ₂ O layer to obtain a 313.8 g. A CH ₂ Cl ₂ layer was subjected to a silica gel column, and a solvent was obtained by using CH ₂ Cl ₂ : MeOH = 20: 1 to 100: 1 and Hexane: EtoAC = 10: 1 to obtain 15.88 g of a single compound. . TLC was performed on this compound. It was confirmed that a single spot having R _f 0.32 was developed in the solvent CH ₂ Cl ₂ : MeOH = 100: 1. Mass and NMR were confirmed to be Weedroll having the molecular formula C ₁₅ H ₂₆ O. Therefore, 15.88 g of active ingredient withol was purified from 10 kg of juniper. The process is shown in FIG.

Example 2 Triglyceride Degradation Effect of Withroll

Triglyceride content was measured to determine the effect of lipolysis on the treatment of 3T3-L1 adipocytes with weedroll.

Specifically, 3T3-L1 adipocytes were dispensed into 12 well plates and incubated for 10 to 12 days to completely differentiate. Once completely differentiated, weedroll was treated by concentration (0, 5, 10, 15, 20, 25, 30 µg / ml) and reacted for 48 hours. After 48 hours, the medium was removed, washed twice with cold PBS (phosphate buffered saline), and treated with 10% formalin and fixed at room temperature for 1 hour. After fixation, formalin was removed, rinsed with 60% isopropanol, and the Oil Red O staining solution (SIGMA-ALDRICH CHEMIE GmbH P.O. 1120, 89552 Steinheim, Germany) was stained at room temperature for 20 minutes. At this time, the Oil Red O staining solution was dissolved 0.7 g Oil Red O in 200ml isopropanol and then diluted to 6: 4 was used after the filter. After staining, the Oil Red O staining solution was removed, washed three times with water, eluted with 100% isopropanol, and the absorbance was measured at 500 nm using a spectrophotometer.

The results are shown in FIGS. 2 and 3. As can be seen in Figures 2 and 3, it was confirmed that the triglyceride content is reduced in a concentration-dependent manner compared to the control treated with 0.5% DMSO, in particular, the triglyceride content was reduced by 31.6% at 30 ㎍ / ㎖. Therefore, weedroll was confirmed to induce lipolysis on fully differentiated adipocytes to decompose triglycerides.

Example  3: Weedroll  Lipolysis ( lipolysis ) effect

When lipolysis is induced in adipocytes, triglycerides are broken down to produce glycerol and fatty acids in the cytoplasm, and when lipolysis occurs continuously, glycerol is released out. Therefore, in order to confirm whether weedol decomposes triglycerides and releases glycerol to induce a lipolytic effect, the amount of glycerol released was measured using a GPO-TRINDER kit after 48 hours of treatment with each concentration.

Specifically, differentiation was induced by the method of Example 2 after dispensing 3T3-L1 adipocytes into 12-well plate. Weed rolls were treated in different concentration-induced adipocytes by concentration and incubated for 48 hours. The amount of glycerol released into the medium was measured using an emzymatic reagent. Glycerol release was measured using glycerol-3-phosphate oxidase (GPO) -TRINDER kit (Sigma). First, Free Glycerol Reagent was dissolved in 40 ml of tertiary sterile water, and then carefully inverted for dissolution. Then, 10 μl of the culture solution treated with Wedroll and 800 μl of Free Glycerol Reagent warmed to 37 ° C. were carefully mixed. And then incubated for 5 minutes at 37 ℃. After incubation, it was measured at 540 nm using a reader (ELISA, Enzyme liked immunosolvant assay).

The results are shown in FIG. As can be seen in Figure 4, it was confirmed that the concentration of glycerol released when treated with the weedroll as compared to the control treated with 0.5% DMSO. In particular, when the 30 ㎍ / ㎖ showed a 2.1 times higher glycerol release than the control group showed a very excellent lipolysis effect.

Example  4 : Weedroll  Differentiation Suppression differentiation inhibition ) effect

When 3T3-L1 adipocytes differentiated into adipocytes, we investigated how weroll affects adipocyte differentiation. Specifically, Oil Red O staining was performed to determine the effect of weedroll differentiation when mouse embryonic 3T3-L1 adipocytes differentiate into adipocytes. First, 3T3-L1 adipocytes were cultured in a 12 well plate and then incubated in a 37%, 5% CO ₂ incubator to be confluent. Incubated for 2 more days in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS and 0.1% gentamycin in a confluent state. Differentiation medium (10% FBS, 0.1% gentamycin, 10µg / ml insulin (SIGMA), 0.5uM dexamethasone, 0.5mM 3-isobuthyl-1 -methylxanthine (Sigma-Aldroch Chemie Gmblt, Riedstr. 2, D-89555, Steinheim, Germany), (MDI)) with Widrol was treated together by concentration. After 3 days of incubation also treated with Weedroll in DMEM medium containing 10㎍ / ㎖ insulin was incubated for 3-5 days. Then, Oil red O staining was performed in the same manner as in Example 6 to measure the content of the triglycerides produced.

The results are shown in FIGS. 5 and 6. As can be seen in Figures 6 and 7, it was confirmed that treatment of the weedrol by concentration than the treatment by only the differentiating agent without processing anything, 100% differentiation significantly inhibited triglyceride accumulation (accumulation). In particular, when the 25 ㎍ / ㎖ was confirmed that the inhibition of triglyceride accumulation 77.6% compared to the control (control). As a result, weedroll not only breaks down triglycerides produced in fully differentiated adipocytes, but also leads to lipolysis, and inhibits adipocyte differentiation by inhibiting the formation of triglycerides when adipocytes differentiate. do.

Example  5: Microarray  analysis

5-1. Experiment method

Using the NimbleGen Whole Oligo 4-plex chip manufactured by JINNO Co., Ltd., the difference in gene expression between 3T3-L1 cells treated with Weedroll and untreated control 3T3-L1 cells was confirmed. The total total RNA sample used in the experiment was carried out after extracting RNA and performing a quality check in the genome. Total RNA samples were subjected to cDNA labeling using fluorescent dyes in the process of synthesizing cDNA through reverse transcription process and hybridized to NimbleGen Mouse Genome Oligo 4-plex chip. NimbleGen Mouse Genome Oligo 4-plex chip used in this experiment was synthesized by repeating 25,631 mouse genes three times using Mouse Genome Build 8 as a database. In the spot synthesized on the NimbleGen Whole Oligo 4-plex chip, a total of 76,083 spots were synthesized at 17.4 mm × 13 mm with the size of 16 μm × 16 μm except for the control gene. The 25,631 oligos contain 2,539 known genes with 23,092 known functions, transcripts and EST sequences that have yet to be identified. In the analysis of DNA chip results, qspline fit and median polish normalization were performed using numerical data of the signal intensity of the spot obtained through scanning. The distribution of all genes obtained as a result of the experiment was displayed as a histogram and compared with the distribution of the standardized data to confirm the accuracy of the experiment.

5-2. Lipolytic effect

In order to elucidate the anti-obesity mechanism of the weed roll, the expression level of mRNA was examined through microarray. Specifically, lipolysis-related genes were compared and analyzed in the weedroll 15 µg / ml treated group and the non-treated group (Table 1.). Adenylate cyclase was increased by +1.38 compared to the control. Adenylate cyclase (Adenylate cyclase) is activated by changing the ATP to cAMP by increasing the concentration of cAMP in the cell can be assumed to activate several genes, such as protein kinase A and protein kinase C there was. In addition, phospholipase β1 (PLβ1), phospholipase A2 (PLA2), phospholipase C β1 (PLC β1), and lipoprotein lipase 1 (LP1) increased by + 2.21, + 1.15, + 3.20, + 1.25, respectively. In addition, ACSL1 (acyl-CoA synthetase long chain family member), a fatty acid transport gene, was found to increase by 2.88. It is believed to transport free fatty acids produced by triglyceride breakdown due to lipolysis. Therefore, weedroll is thought to cause lipolysis by activating adenylate cyclase to increase the concentration of cAMP in the cells as well as activating various lipases to break down triglycerides.

Gene Gene Bank No . Intensity ratio Explanation AC 8 NM_009623 + 1.38 Adenylate cyclase 8 PL _β1 XM_979248 + 2.21 Phospholipase β1 PLA ₂ NM_00869 + 1.15 Phospholipase A2 PLC _β1 NM_019677 + 3.20 Phospholipase C β1 LP 1 NM_008509 + 1.25 Lipoprotein lipase 1 ACSL1 NM_007981 + 2.88 Acyl-CoA synthetase long chain family member, ACSL1

5-3. Differentiation inhibitory effect

The expression level of adipocyte differentiation-related mRNA was compared (Table 2). C / EBP _β (CCAAT enhancer binding protein beta), which is activated at the early stage of adipocyte differentiation, that is, during clonal expansion, was reduced by -0.31. (CCAAT enhancer binding protein gamma) was also reduced by -0.23, confirming that weedroll inhibits adipocyte differentiation. In addition, the lipogenesis gene SCD-1 (stearoyl-Coenzyme A desaturase 1)-0.62 was also found to be markedly reduced. These results suggest that weedroll inhibits the differentiation of adipocytes by inhibiting the clonal expansion stage, which is the early stage of adipocyte differentiation.

Gene Gene Bank No . Expression fold Explanation PPARα NM_011144 -0.28 Peroxisome proliferator
activated receptor, alpha C / EBPβ NM_009883 -0.31 CCAAT / enhancer binding
protein (C / EBP), beta C / EBPγ NM_009884 -0.23 CCAAT / enhancer binding
protein (C / EBP), gamma SCD-1 NM_009127 -0.62 Stearoyl-coenzyme a
desaturase 1

Example  6: Weston Blot  analysis

After collecting 3T3-L1 cells treated with wedrol at different concentrations (0, 1, 5, 10, 15, 20 µg / ml), washed once with PBS, and then washed with CSK buffer (100 mM PIPES (pH6.8), 100 mM). Suspension was dissolved in NaCl, 1 mM MgCl ₂ , 1 mM EGTA, 1 mM dithiothreitol (DTT) and 1 mM EGTA using 0.1% Triton X-100, 1 mM ATP and Protease inhibitor Cocktail (BD Pharmingen), and dissolved for 15 minutes. 14,000 rpm was centrifuged for 20 minutes. The supernatant was again suspended in CSK buffer and disrupted by sonication. The crushed cells were centrifuged at 14,000 rpm for 20 minutes, the supernatant was quantified by BCA method, and 30-40 ㎍ protein was electrophoresed on SDS-PAGE gel. After electrophoresis, the protein in the gel was transferred to the PVDF membrane and blocked with 5% skim milk at 4 ° C. for 16 hours (O / N). The primary antibody was reacted at 4 ° C. for 16 hours (O / N) and the membrane was washed with TBS (50 mM Tris-HCl (pH7.5) and 0.15M NaCl) with 0.1% Triton X-100 solution. Next, the secondary antibody was added and reacted for 16 hours (O / N). After washing the membrane, the immunoreactive protein was detected by a Chemi-luminescence system (SuperSignal West Femto Maxium sensitivity Substrate, Pierce). Antibodies used in this experiment were Santa Cruz Biothechnology Inc. And Cell signaling technology.

In general, adipocyte differentiation is related to the expression of transcription factors acting on the regulatory regions of genes that are characteristic of adipocytes in the process of adipocyte proliferation into adipocytes. Transcription factors involved in this process include peroxisome proliferation activated receptor gamma (PPAR), CCAAT enhancer binding protein (C / EBP), and E2F transcription factor 1 (E2F 1). Induced differentiators promote the expression of these transcription factors to convert adipocytes into adipocytes. Inhibiting the expression of these enzymes inhibits the conversion to adipocytes and reduces the accumulation of fat.

As shown in Figure 7, when the treatment with the weed was confirmed that inhibiting the conversion to adipocytes and inhibited differentiation by reducing the PPAR concentration-dependently. In addition, it was confirmed that the inhibition of adipocyte differentiation by inhibiting cell expansion by inhibiting cell division by reducing E2F 1, which promotes cell division in cell growth and various differentiation models. In addition, compared to pRb, the activated phospho-pRb was found to be significantly reduced when weedroll was treated, indicating that E2F 1 and pRb-binding forms were phosphorylated more than E2F 1 activated.

In addition, differentiation into adipocytes is activated E2F 1 by cyclin dependent kinase (CDK) induced by C / EBPβ, C / EBPδ activated during clonal expansion. It was confirmed that p21, a CDK inhibitor, was increased in a concentration-dependent manner when treated with Weedroll. Therefore, weedroll seems to inhibit the differentiation of adipocytes by inhibiting the time of clonal expansion.

Example  7: Acute Toxicity Test

Mice (20 ± 5 g, central experimental animals) and rats (235 ± 10 g, central experimental animals) were used for the acute toxicity test of the compound of the present invention, Widrol. ICR-based mice and Sprague-Dawley rats were divided into four groups of 10 animals each, and the juniper extract and compound of the present invention were orally administered at a dose of 100 and 250 mg / kg, respectively. No deaths occurred in all cases and no symptoms were apparent from the control group.

Weedroll or a pharmaceutically acceptable salt thereof of the present invention is excellent in lipolytic effect and the effect of inhibiting the differentiation of adipocytes into adipocytes can be useful for the prevention or treatment of obesity.

1 is a diagram illustrating a process for separating the withroll compound from the juniper extract.

Figure 2 is a picture of measuring the triglyceride content in the cells by Oil Red O staining when treated with a concentration of the precursor compound of the present invention to 3T3-L1 fat cells.

Figure 3 is a graph showing the results of observing the triglyceride content in the cells when treated with a concentration of the precursor compound of the present invention to 3T3-L1 fat cells.

Figure 4 is a diagram showing the results of observing the amount of glycerol released in the cells when treated with the concentration of the precursor compound of the present invention to 3T3-L1 fat cells.

FIG. 5 shows triglycerides in cells when the precursor cells of the present invention were treated with concentrations of the precursor compounds of the present invention in 3T3-L1 adipocytes in order to confirm the inhibitory effect of the present invention, the adipocytes differentiate into adipocytes. This figure is measured by Oil Red O staining.

Figure 6 is a triglyceride in the cells when treated with a concentration of the precursor precursor of the present invention to 3T3-L1 fat precursor cells in order to confirm the inhibitory effect of the weedroll compound of the present invention when adipocytes differentiate into adipocytes The figure shows the result of observing the accumulation amount.

Figure 7 is a graph showing the results of analysis of Weston blot the expression level of the proliferation-related protein differentiation of adipocytes into adipocytes when treated with the concentration of the wedrol compound of the present invention to the 3T3-L1 adipocytes.

Claims (4)

A pharmaceutical composition for the prevention and treatment of obesity, including widrol or a pharmaceutically acceptable salt thereof. The pharmaceutical composition of claim 1, wherein the weedroll or a pharmaceutically acceptable salt thereof is 0.1-50% by weight of the total weight of the composition. The pharmaceutical composition of claim 1, wherein the weedroll is from juniper. Health functional food for the prevention of obesity, including widrol or a pharmaceutically acceptable salt thereof.

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