KR20220086993A - Composition for preventing or treating fatty liver or metabolic syndrome containing thymol derived from turmeric as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing or treating fatty liver or metabolic syndrome comprising thymol derived from turmeric as an active ingredient. , reduces fat accumulation without cytotoxicity, reduces triglycerides and total cholesterol, and effectively inhibits the expression of genes and proteins of SREBP-1c, ACC, FAS, C/EBP and PPAR associated with fat accumulation, and , it was confirmed that increasing AMPK, a fatty acid oxidation factor, inhibited fat accumulation and improved fatty liver or metabolic syndrome.

Description

Composition for preventing or treating fatty liver or metabolic syndrome containing thymol derived from turmeric as an active ingredient {Composition for preventing or treating fatty liver or metabolic syndrome containing thymol derived from turmeric as an active ingredient}

The present invention relates to a composition for preventing or treating fatty liver or metabolic syndrome comprising thymol derived from turmeric as an active ingredient.

Fatty liver disease, also called fatty liver, is a disease that causes liver damage due to abnormal accumulation of fat (triglycerides, etc.) in liver cells. It is known that the initial condition of fatty liver disease is simple fatty liver in which only fat deposition is recognized in hepatocytes, and then the condition progresses to steatohepatitis (including liver fibrosis), and cirrhosis or hepatocellular carcinoma. In general, causes of fat deposition in the liver include alcohol intake, obesity, diabetes, abnormal lipid metabolism, drugs (steroids, tetracycline, etc.), Cushing's syndrome, poisoning (eg yellow phosphorus), and severe nutritional disorders.

The causes of fatty liver disease are largely divided into alcoholic and non-alcoholic. A liver disease caused by the former is alcoholic fatty liver disease (also called alcoholic liver disease), and a liver disease caused by the latter is non-alcoholic fatty liver disease ( It is called nonalcoholic fatty liver disease (NAFLD).

Alcoholic fatty liver disease progresses from simple fatty liver in the early stage to steatohepatitis and cirrhosis.

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease, and is characterized by the accumulation of triglycerides in the liver cells of patients without excessive alcohol intake. The prevalence of nonalcoholic fatty liver disease continues to increase in parallel with the overnutrition associated with high-fat and high-carbohydrate intake.

Nonalcoholic fatty liver disease patients usually have obesity, diabetes, and hyperlipidemia, but not all obese people develop nonalcoholic fatty liver disease, and various factors may play an important role in the development of nonalcoholic fatty liver disease. In particular, nonalcoholic fatty liver disease is divided into primary and secondary according to the cause. bypass surgery), various drugs (glucocorticoids, estrogens, tamoxifen, methotrexate, zidovudine, amiodarone, tetracycline, didanosine, cocaine, diltiazem, perhexiline), toxic substances (poisonous mushrooms, bacterial toxins), metabolic causes (lipodystrophy, dysbetalipoporoteinemia, Weber-Christian syndrome) It is known to be caused by Wolman's disease, acute fatty liver of pregnancy, Reye's syndrome) and other factors (inflammatory bowel syndrome, AIDS infection: HIV infection).

In addition, liver accumulation of triglycerides directly related to non-alcoholic fatty liver disease and hepatocellular damage that progresses therefrom are caused by changes in systemic factors such as local factors and insulin resistance to influx/synthesis of lipids into the liver and release/oxidation imbalance. That is, it is known that fatty acids are introduced into the liver in excess of fatty acids that can be oxidized by mitochondria of hepatocytes due to insulin hyperinsulinemia caused by insulin resistance, and triglycerides are accumulated in the liver. The expression of lipogenic transcription factors, peroxisome proliferator-activiated receptor-gamma (PPAR-γ) and sterol regulatory element binding protein-1c (SREBP-1c), is increased (upregulation) by insulin resistance, resulting in lipogenesis in the liver. An increase in synthesis (de novo hepatic lipogenesis) may also result in the accumulation of triglycerides.

On the other hand, the worldwide frequency of the incidence of nonalcoholic fatty liver is 9-37%, and the prevalence of obesity and diabetes in the Asia-Pacific region is 15-21%, and it is gradually increasing.

Accordingly, despite the beneficial and therapeutic effects of antioxidants and insulin sensitizers such as metformin and thiazolidinedione, body control through lifestyle changes is the basis for the treatment of NAFLD. recognized as effective and effective. Therefore, there is a lot of interest in natural products that effectively increase fat oxidation, reduce lipid production and regulate lipid metabolism.

Metabolic syndrome is characterized by high levels of blood fat, hypertension, insulin resistance and central obesity (excess adipose tissue in the abdominal region), which underlies and causes all geriatric diseases namely obesity, hypertension, hyperlipidemia, and hyperglycemia. becomes this Metabolic syndrome is a phenomenon in which dangerous adult diseases such as arteriosclerosis, high blood pressure, obesity, diabetes, and hyperlipidemia occur simultaneously in one person. Studies on metabolic syndrome such as obesity, arteriosclerosis, diabetes, and hyperlipidemia are continuously being conducted.

Turmeric ( Curcuma Longae ) is a perennial plant classified in the genus Curcuma of the ginger family ( Zingiberaceae ), is native to tropical Asia, is cultivated in India, China, and Southeast Asia, and is used as food, medicinal, and natural dyes. Recently, it has been reported in various papers and media that curcuminoids and curcumin have various physiological effects such as antioxidant, anti-cancer, anti-mutation, anti-inflammatory, dementia prevention, liver function protection, and cholesterol reduction.

An object of the present invention is to provide a health functional food composition for preventing or improving fatty liver or metabolic syndrome comprising thymol as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating fatty liver or metabolic syndrome comprising thymol as an active ingredient.

Another object of the present invention is to prepare an extract by 1) extracting the dried turmeric with a solvent;

2) preparing a fraction by adding a fractionation solvent to the extract of step 1); and

3) It is to provide a method for preparing a health functional food composition for preventing or improving fatty liver or metabolic syndrome comprising the step of purifying thymol derived from turmeric from the fraction of step 2).

Another object of the present invention is to prepare an extract by 1) extracting the dried turmeric with a solvent;

2) preparing a fraction by adding a fractionation solvent to the extract of step 1); and

3) To provide a method for preparing a pharmaceutical composition for the prevention or treatment of fatty liver or metabolic syndrome, comprising the step of purifying thymol derived from turmeric from the fraction of step 2).

The present invention provides a health functional food composition for preventing or improving fatty liver or metabolic syndrome comprising thymol as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating fatty liver or metabolic syndrome comprising thymol as an active ingredient.

In addition, the present invention comprises the steps of 1) extracting the dried turmeric with a solvent to prepare an extract;

2) preparing a fraction by adding a fractionation solvent to the extract of step 1); and

3) It provides a method for preparing a health functional food composition for preventing or improving fatty liver or metabolic syndrome, comprising the step of purifying thymol derived from turmeric from the fraction of step 2).

In addition, the present invention comprises the steps of 1) extracting the dried turmeric with a solvent to prepare an extract;

2) preparing a fraction by adding a fractionation solvent to the extract of step 1); and

3) It provides a method for preparing a pharmaceutical composition for preventing or treating fatty liver or metabolic syndrome, comprising the step of purifying thymol derived from turmeric from the fraction of step 2).

When thymol derived from turmeric of the present invention is treated with oleic acid-induced HepG2 liver cancer cell line, it reduces fat accumulation without cytotoxicity, reduces triglycerides and total cholesterol, and SREBP associated with fat accumulation -1c, ACC, FAS, C/EBP and PPAR genes and protein expression effectively suppressed, and fatty acid oxidation factor, AMPK, was confirmed to be increased, thereby inhibiting fat accumulation and improving fatty liver or metabolic syndrome Therefore, it can be usefully used in related industries.

Figure 1a is a diagram showing the chemical formula and high-performance liquid chromatography (HPLC) results of thymol separated from the fraction fractionated with turmeric 80% methanol extract, Figure 1b is HPLC of turmeric 50% fermented ethanol extract A diagram showing the results.
Figure 2 is a view confirming the cell viability by treating the turmeric-derived thymol (thymol, THY) of the present invention in HepG2 cell line.
3 is a diagram illustrating the inhibition of fat accumulation by treatment with oleic acid (oleate, OA) in a HepG2 cell line induced to accumulate fat by staining with Oil Red O (a: control, b: oleic acid alone treatment group, c: turmeric extract treatment group, d: positive control group, e: thymol 100 μM treatment group, f: thymol 200 μM treatment group).
Figure 4 is treated with turmeric-derived thymol (THY) of the present invention in the HepG2 cell line induced to accumulate fat with oleic acid (oleate, OA), triacylglycerol (triacylglycerol, triglyceride, TG) and total cholesterol, TC) is a diagram confirming inhibition.
Figure 5 is a result of quantitative-real-time RT-PCR analysis of the expression of genes related to fat accumulation by treating the turmeric-derived thymol (THY) of the present invention in the HepG2 cell line induced to accumulate fat with oleic acid (oleate, OA). (a: SRERP-1c expression confirmation, b: FAS expression confirmation, c: ACC expression confirmation, d: C/EBP expression confirmation, e: PPARγ expression confirmation).
6 shows the results of analyzing the expression of proteins related to fat accumulation by Western blot by treating the turmeric-derived thymol (THY) of the present invention with oleic acid (oleate, OA) to the HepG2 cell line induced to accumulate fat.
Figure 7 is a fat oxidizing factor AMP-activated protein kinase (AMPK) by treating the turmeric-derived thymol (THY) of the present invention with oleic acid (oleate, OA) in the HepG2 cell line induced to accumulate fat. is the result of analysis of phosphorylation by Western blot.

The present invention provides a health functional food composition for preventing or improving fatty liver or metabolic syndrome comprising thymol as an active ingredient.

As used herein, the term “prevention” refers to any action that suppresses the symptoms of a specific disease or delays the progression by administration of the composition of the present invention.

As used herein, the term “improvement” refers to any action that at least reduces a parameter related to a condition to be treated, for example, the severity of symptoms.

The “fatty liver” or “fatty liver disease” of the present invention is an indication for the accumulation of excess fat (triglyceride) in the liver. When more than 5% of the liver weight is accumulated, fatty liver is diagnosed. It is divided into alcoholic fatty liver and nonalcoholic fatty liver caused by factors such as hyperlipidemia, diabetes and obesity.

According to an embodiment of the present invention, the fatty liver may be selected from the group consisting of alcoholic fatty liver, non-alcoholic fatty liver, nutrient fatty liver, starvation fatty liver, obese fatty liver and diabetic fatty liver, and preferably non-alcoholic fatty liver. Sexual fatty liver, but is not limited thereto.

According to one embodiment of the present invention, the metabolic syndrome may be selected from the group consisting of obesity, dyslipidemia, hyperlipidemia, diabetes and insulin resistance syndrome, preferably obesity or hyperlipidemia, but is not limited thereto.

In addition to containing the active ingredient of the present invention, the food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional food composition.

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 such as conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The above-mentioned flavoring agents can advantageously use natural flavoring agents (Taumatine), stevia extract (eg rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention may be formulated in the same manner as the 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, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements. There is this.

In addition, the food composition includes, in addition to the active ingredient extract, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, 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 beverages and vegetable beverages.

The functional food composition of the present invention may be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing or treating fatty liver or metabolic syndrome. In the present invention, the term 'health functional food composition' refers to food manufactured and processed using raw materials or ingredients useful for the human body according to Act No. 6727 of the Health Functional Food Act, and It refers to ingestion for the purpose of obtaining useful effects for health purposes such as regulating nutrients or physiological effects. The health functional food of the present invention may contain normal food additives, and unless otherwise specified, whether it is suitable as a food additive is related to the item according to the general rules and general test method of food additives approved by the Food and Drug Administration. It is judged according to the standards and standards. The items listed in the 'Food Additives Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high pigment, and guar gum; Mixed preparations, such as a sodium L-glutamate preparation, a noodle-added alkali agent, a preservative preparation, and a tar color preparation, etc. are mentioned. For example, a health functional food in tablet form is granulated by a conventional method by mixing a mixture of the active ingredient of the present invention with an excipient, binder, disintegrant and other additives, followed by compression molding by putting a lubricant, etc., or The mixture can be compression molded directly. In addition, the health functional food in the form of tablets may contain a corrosive agent and the like, if necessary. Among health functional foods in capsule form, hard capsules can be prepared by filling a mixture of the active ingredient of the present invention with additives such as excipients in a conventional hard capsule. It can be prepared by filling the mixture mixed with the capsule base such as gelatin. The soft capsules may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary. A health functional food in the form of a ring can be prepared by molding a mixture of the active ingredient of the present invention with an excipient, a binder, a disintegrant, etc. by a known method, Alternatively, the surface may be coated with a material such as starch or talc. The health functional food in the form of granules can be prepared in granular form by a conventionally known method by mixing a mixture of the active ingredient excipients, binders, disintegrants, etc. of the present invention, and may contain flavoring agents, flavoring agents, etc. as needed can

According to an embodiment of the present invention, the thymol has a structure of Formula 1 below.

[Formula 1]

According to an embodiment of the present invention, the thymol may be separated from the turmeric extract, and may be extracted with a solvent selected from the group consisting of water, C1 to C4 lower alcohols and aqueous solutions of lower alcohols, preferably methanol or Ethanol, but not limited thereto.

According to an embodiment of the present invention, the thymol may be separated from the fraction of the turmeric extract, and the fraction of the turmeric extract is selected from the group consisting of water, C1 to C4 lower alcohol, aqueous lower alcohol solution, chloroform and ethyl acetate. It may be fractionated with a solvent, preferably an ethyl acetate fraction, but is not limited thereto.

According to an embodiment of the present invention, the thymol is a fat accumulation factor, sterol regulatory element-binding protein-1c (SREBP-1c), acetyl-CoA carboxylase (acetyl-CoA carboxylase, ACC), fatty acid synthase (FAS), CCAAT-enhancer-binding protein (C/EBP), and proliferator-activated receptor γ (PPARγ) selected from the group consisting of It may be to inhibit the expression of a gene or protein.

According to an embodiment of the present invention, the thymol may increase phosphorylation of AMP-activated protein kinase (AMPK), which is a fat oxidation factor.

In addition, the present invention provides a pharmaceutical composition for preventing or treating fatty liver or metabolic syndrome comprising thymol as an active ingredient.

As used herein, the term “treatment” refers to any action that improves or beneficially changes the symptoms of a specific disease by administration of the composition of the present invention.

The pharmaceutical composition of the present invention may further include an adjuvant in addition to the active ingredient. The adjuvant may be used without limitation as long as it is known in the art, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to increase the effect.

The pharmaceutical composition according to the present invention may be prepared in a form in which the active ingredient is incorporated into a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention may be formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injection solutions according to conventional methods, respectively. .

In the case of formulation, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient in the active ingredient, for example, starch, calcium carbonate, sucrose, lactose, gelatin. It can be prepared by mixing and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid formulations for oral administration include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. can Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As the base of the suppository, witepsol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The pharmaceutical composition according to the present invention may be administered to an individual by various routes. Any mode of administration can be envisaged, for example, by oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.

The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the individual's age, weight, sex, physical condition, and the like. It is self-evident that the concentration of the active ingredient included in the pharmaceutical composition can be variously selected depending on the subject, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. If the concentration is less than 0.01 μg/ml, pharmaceutical activity may not appear, and if it exceeds 5,000 μg/ml, it may be toxic to the human body.

In addition, the present invention comprises the steps of 1) extracting the dried turmeric with a solvent to prepare an extract;

2) preparing a fraction by adding a fractionation solvent to the extract of step 1); and

3) It provides a method for preparing a health functional food composition for preventing or improving fatty liver or metabolic syndrome, comprising the step of purifying thymol derived from turmeric from the fraction of step 2).

In addition, the present invention comprises the steps of 1) extracting the dried turmeric with a solvent to prepare an extract;

2) preparing a fraction by adding a fractionation solvent to the extract of step 1); and

3) It provides a method for preparing a pharmaceutical composition for preventing or treating fatty liver or metabolic syndrome, comprising the step of purifying thymol derived from turmeric from the fraction of step 2).

Hereinafter, the present invention will be described in more detail by way of Examples. These examples are merely for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited to these examples.

<Example 1> Separation of thymol derived from turmeric

<1-1> Extraction method and separation method

In order to separate thymol (Thymol, THY) derived from turmeric of the present invention, dried and sliced turmeric was extracted twice under reflux at room temperature with an 80% aqueous methanol solution as a solvent. The extracted turmeric extract was fractionated in the order of water (1 L), ethyl acetate (EtOAc 1L, 3 times), and n-butanol (0.8 L, 3 times). 30 g of the ethyl acetate fraction (CLE) was taken from the fraction, and dissolved in a solvent in which chloroform-methanol (CHCL ₃ -MeOH) was mixed in a ratio of 100: 1, 50: 1, 30: 1, and 10: 1, respectively, followed by thin layer chromatography By monitoring (thin-layer chromatography, TLC), 10 fractions (CLE-1 to CLE-10) were obtained. CLE-2 (elution volume/total volume [Ve/Vt], 0.21-0.28; 900 mg) of the fraction was filled in a SiO ₂ column, and CHCL ₃ -MeOH (50: 1, 2L) was added as a solvent. fractionation to obtain CLE-2-1 to CLE-2-5 fractions. The obtained CLE-2-2 fraction was filled in an octadecyl silica gel (ODS) column, and finally thymol was separated using MeOH-H ₂ O (10: 1, 1 L) as a solvent, and the separated broad-spectrum The structure of the compound was determined based on the spectroscopic data (FIG. 1A).

<1-2> High-Performance Liquid Chromatography Analysis (HPLC) analysis

In order to confirm the yield of thymol in the turmeric extract (CE), high-performance liquid chromatography (HPLC) analysis was performed. Specifically, dried and sliced turmeric was homogenized using a ball mill (Ball mill, Retsch MM400, Haan, Germany). The homogenized turmeric powder was sieved through a 40 mesh sieve, and 1000 mg of the powder was ultrasonically extracted at 35° C. for 1 hour by adding 5 ml of 50% fermented ethanol (EtOH). The extracted extract was filtered using a syringe filter (0.22 μm), the solvent of the filtrate was evaporated under reduced pressure, and the extracted extract was directly injected into the HPLC system. HPLC was performed using a 1200-RRLC system (Agilent Technologies, Palo Alto, CA, USA) equipped with a binary solvent delivery system and autosampler. Specifically, chromatography was performed on a YMC Pack ODS-AM column (4.6250 mm, 5 m), the column oven was maintained at 35° C., and the mobile phase was solvent A (0.1 % [v/v] aqueous formic acid) and solvent B (0.1 % [v/v] of acetonitrile containing formic acid) was used. As the optimal conditions for HPLC, analysis was performed at a flow rate of 1 ml/min under the conditions of 0 to 3 minutes 70% solvent B, 3 to 16 minutes 50% solvent B, 16 to 20 minutes 30% solvent B, and then an automatic sample injector. was used to inject 10 μL of solvent A. Finally, it was confirmed that the yield of thymol (THY) isolated from the 50% fermented ethanol extract was 10.67 mg/g (FIG. 1B).

<Example 2> Confirmation of improvement of fatty liver

<2-1> Cell culture

In order to confirm the improvement effect of turmeric-derived thymol of the present invention on nonalcoholic fatty liver, the human liver cancer cell line HepG2 cell line was purchased from American Tissue Culture Collection (ATCC, catalog no. HB-8065; Manassas, VA, USA), and DMEM and 10% fetal bovine serum. Culture conditions were 95% humidity, 5% CO ₂ and cultured at 37°C.

<2-2> Cell viability analysis

To evaluate the cytotoxicity of thymol, a cell viability assay was performed. Specifically, MTS analysis was performed, and the HepG2 cell line cultured in Example 2-1 was 5 x 10^5 cells in 24-well cell culture plates (24-well cell culture plates, Nunc, A/S, Roskilde, Denmark). It was inoculated into each well to achieve density and then cultured for 24 hours. After incubation, each well was treated with 200 μg of turmeric extract (CE), 20 μg of positive control (Silymarin, SM), 50, 100, 200 and 400 μM of thymol (THY), and oleic acid (oleate, OA) to induce fat accumulation. ) was added to 500 μM, and cultured for 18 hours (5% CO ₂ , 37 ° C.), and after incubation, the existing medium was replaced with a medium with MTS solution (5 mg/ml) added, and after 20 minutes, a microplate reader ( Using a microplate reader, BioTek South Korea, Seoul, Korea), the absorbance of each well was measured at 490 nm.

As a result, as shown in Figure 2, even if the treatment concentration of thymol (THY) derived from turmeric increased, it did not affect the cell viability, it was confirmed that there is no cytotoxicity.

<2-3> Fat accumulation analysis

For fat accumulation analysis, the HepG2 cell line cultured in Example 2-1 was inoculated to a concentration of 4 x 10^5 cells/ml in a 24-well plate, turmeric extract (CE) 200 μg, positive control (silymarin, Silymarin, SM) 20 μg, thymol (THY) 100 and 200 μM were treated, and 500 μM of oleic acid (oleate, OA) was added to induce fat accumulation, and cultured for 24 hours. The cultured cells were washed with cold DPBS and fixed with 10% paraformaldehyde for 1 hour. The fixed cells were washed again with DPBS and finally with 60% isopropanol. In order to observe the fat accumulated in the cells after washing, the cells were stained with Oil Red O (Sigma-Aldrich, St. Louis, MO, USA) for 1 hour, and after removing the remaining dye with 60% isopropanol, each well was optically It was observed under a microscope.

As a result, as shown in FIG. 3 , it was confirmed that, in the HepG2 cell line, which is a hepatocyte, treatment with oleic acid induced fat accumulation. Confirmed.

<Example 3> Lipid metabolism analysis

In order to determine whether thymol derived from turmeric affects the metabolism of total cholesterol (TC) and triacylglycerol (TG, neutral fat), which are indicators of lipid metabolism, total cholesterol and triacylglycerol were measured. Specifically, HepG2 cells were inoculated at a concentration of 4 x 10^5 cell/ml in a 24-well cell culture plate, and cultured for 24 hours. Cultured cells were treated with 200 μg of turmeric extract (CE), 20 μg of positive control (SM), 100 and 200 μM of thymol (THY), and 500 μM of oleic acid (oleate, OA) were treated together to induce fat accumulation 24 time incubation. After incubation, the levels of TC and TG were measured according to the TC and TG measurement kit protocol, and after the end of the experiment, the absorbance of each well was measured as absorbance at 450 nm using a microplate reader.

As a result, as shown in FIG. 4, when the HepG2 cell line was treated with oleic acid, it was confirmed that triacylglycerol (TG) and total cholesterol (TC) increased, and when treated with thymol derived from turmeric, triacyl It was confirmed that the production of glycerol and total cholesterol was reduced.

<Example 4> Quantitative real time reverse transcription PCR (q real-time RT-PCR) analysis

To determine the effect of turmeric-derived thymol of the present invention on the expression change of the fat metabolism gene, the expression of the fat metabolism gene was analyzed. Specifically, HepG2 cells were inoculated at a concentration of 4 x 10^5 cell/ml in a 24-well cell culture plate, and cultured for 24 hours. Cultured cells were treated with 200 μg of turmeric extract (CE), 20 μg of positive control (SM), 100 and 200 μM of thymol (THY), and 500 μM of oleic acid (oleate, OA) were treated together to induce fat accumulation 24 time incubation. Total RNA was extracted from the cultured cells using the E.Z.N.A.® Total RNA Kit (Omega Bio-tek, Inc.; Norcross, GA, USA) according to the manufacturer's protocol. Reverse transcription was performed for transcription analysis, and reverse transcription was performed using a QuantiTect® Reverse Transcription Kit (Qiagen, Hilden, Germany), and quantitative real-time reverse transcription PCR was performed using Power SYBR® Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). Foster City, CA, USA). Fat accumulation-related genes sterol regulatory element-binding protein-1c (SREBP-1c), acetyl-CoA carboxylase (ACC), fatty acid synthase (fatty acid synthase, FAS), CCAAT-enhancer-binding protein (CCAAT-enhancer-binding protein, C/EBP) and primers for expression analysis of proliferator-activated receptor γ (PPARγ) are shown in Table 1 below.

target gene Primer sequence (5'-3') forward reverse SREBP-1c CGC AAG GCC ATC GAC TAC AT GAC TTA GGT TCT CCT GCT TGA GTT TC FAS AAG GAC CTG TCT AGG TTT GAT GC TGG CTT CAT AGG TGA CTT CCA ACC TCG CTT TGG GGG AAA TAA AGT G ACC ACC TAC GGA TAG ACC GC C/EBP CAA GCA CAG CGA CGA GTA CAA GCT TGA ACA AGT TCC GCA GGG PPARγ GCA GGC TCC ACT TTG ATT ACC ACT CCC ACT CCT TTG GAPDH CAG GGC TGC TTT TAA CTC TGG T GAT TTT GGA GGG ATC TCG CT

As a result, as shown in FIG. 5, the expression of SREBP-1c, ACC, FAS, C/EBP and PPARγ, which are genes related to fat accumulation, increased when oleic acid was treated in HepG2 cell line, and thymol derived from turmeric was concentration-dependent when treated. As a result, it was confirmed that the expression of SREBP-1c, ACC, FAS, C/EBP and PPARγ related to fat accumulation was decreased.

<Example 5> Western blot analysis

In Example 4, Western blot analysis was performed to determine whether thymol-derived turmeric inhibited not only the expression of genes related to fat accumulation, but also protein expression. Specifically, HepG2 cells were inoculated into a 6-well plate at a concentration of 5 x 10^5 cell/ml. After inoculation, cells were treated with 20 μg of positive control (SM), and 100 and 200 μM of thymol derived from turmeric (THY). To induce fat accumulation 1 hour after treatment, oleic acid (oleate, OA) was treated at a concentration of 500 μM and supplemented with 95% humidity and 5% CO ₂ , and cultured at 37° C. for 24 hours. After incubation, the cells were washed twice with DPBS, and the cells were lysed with a cell lysis buffer for 2 hours. Lysed cells were centrifuged at 16,000 g for protein extraction at 4°C for 5 minutes. The obtained protein was quantified according to the quantification protocol, and the quantified sample was subjected to SDS gel electrophoresis. The electrophoresed protein was transferred to a polyvinylidene difluoride membrane under the condition of 15V for 45 minutes. The membrane was blocked with 3% BSA for 1 h, and SREBP-1c (1 : 1000), FAS (1 : 1000), ACC (1 : 1000), C/EBP (1 : 1000), PPAR (1 : 1000) and A primary antibody against pAMPK (1:1000) was used to react at 4 °C for 18 h. After the reaction, the membrane was washed several times with TBST and treated with each secondary antibody (1:5000) at room temperature. After treatment, the membrane was washed with TBST, the washed membrane was analyzed using Atto ECL plus (Tokyo, Japan), and an Image Quant LAS 4000 Mini Bio molecular imager (GE Healthcare, UK) was used for band analysis.

In addition, the expression and phosphorylation of AMP-activated protein kinase (AMPK), which is a major regulator of adipogenesis and fatty acid oxidation in metabolic tissues, was further confirmed using the above Western blot method, and AMPK (1 : 1000) was used, and the secondary antibody (1 : 5000) was used.

Primary antibodies to FAS, SREBP-1c, PPAR and control (β-actin, house keeping gene) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), and ACC, P-AMPK, AMPK 1 The primary antibody was purchased from Cell Signaling (Danvers, MA, USA), and a mouse or rabbit-derived antibody was used as the primary antibody. As the secondary antibody, goat-anti-mouse IgG (Goat Anti-Mouse IgG) or goat-anti-rabbit IgG (Goat Anti-Rabbit IgG) was used.

As a result, as shown in FIG. 6 , it was confirmed that the protein expression of SREBP-1c, ACC, FAS, C/EBP and PPAR related to fat accumulation increased when oleic acid was treated in the HepG2 cell line, and thymol derived from turmeric ( THY), it was confirmed that the protein expression of SREBP-1c, ACC, FAS, C/EBP and PPAR was decreased in a concentration-dependent manner.

In addition, as shown in FIG. 7 , as a result of confirming phosphorylation of AMPK, it was confirmed that phosphorylation of AMPK was increased when treated with thymol (THY) derived from turmeric, compared to HepG2 cells induced by oleic acid to accumulate fat.

<Example 6> Statistical analysis

Data of all examples of the present invention were collected for statistical analysis, and various comparison tests of Duncan were performed. All examples were performed three times, and the mean and standard deviation of three independent experiments were described. Statistical analysis was performed using SPSS Statistics 23 software (SPSS Inc., Chicago, IL, USA), and p<0.05 (indicated by *) and p<0.01 (indicated by **) were considered statistically significant. did

Therefore, when thymol derived from turmeric of the present invention is treated with oleic acid-induced HepG2 liver cancer cell line, it reduces fat accumulation without cytotoxicity, reduces triglycerides and total cholesterol, and reduces fat accumulation and By effectively inhibiting the expression of related SREBP-1c, ACC, FAS, C/EBP and PPAR genes and proteins, and increasing AMPK, a fatty acid oxidizing factor, it was confirmed that fatty liver or metabolic syndrome was improved.

Claims (24)

A health functional food composition for preventing or improving fatty liver or metabolic syndrome comprising thymol as an active ingredient. The method of claim 1,
The thymol is a health functional food composition for preventing or improving fatty liver or metabolic syndrome, characterized in that it is represented by Formula 1
[Formula 1]

. 3. The method of claim 2,
The thymol is a health functional food composition for preventing or improving fatty liver or metabolic syndrome, characterized in that it is separated from turmeric ( Curcuma Longae ) extract. 4. The method of claim 3,
The turmeric extract is a health functional food composition for preventing or improving fatty liver or metabolic syndrome, characterized in that it is extracted with a solvent selected from the group consisting of water, C1 to C4 lower alcohols and aqueous solutions of lower alcohols. 3. The method of claim 2,
The thymol is a health functional food composition for the prevention or improvement of fatty liver or metabolic syndrome, characterized in that it is separated from the fraction of the turmeric extract. 6. The method of claim 5,
The fraction is water, C1 to C4 lower alcohol, aqueous solution of lower alcohol, fatty liver or a health functional food composition for the prevention or improvement of metabolic syndrome, characterized in that fractionated with a solvent selected from the group consisting of chloroform and ethyl acetate. 7. The method of claim 6,
The fraction is a health functional food composition for preventing or improving fatty liver or metabolic syndrome, characterized in that it is further fractionated from the ethyl acetate fraction. The method of claim 1,
The thymol is a fat accumulation factor, sterol regulatory element-binding protein-1c (SREBP-1c), acetyl-CoA carboxylase (acetyl-CoA carbo xylase, ACC), fatty acid synthase ( Expression of a gene or protein selected from the group consisting of fatty acid synthase (FAS), CCAAT-enhancer-binding protein (C/EBP), and proliferator-activated receptor γ (PPARγ) A health functional food composition for preventing or improving fatty liver or metabolic syndrome, characterized in that it inhibits. The method of claim 1,
The thymol is a fatty oxidizing factor AMP-activated protein kinase (AMP-activated protein kinase, AMPK ) A health functional food composition for the prevention or improvement of fatty liver or metabolic syndrome, characterized in that it increases phosphorylation. The method of claim 1,
Wherein the fatty liver is selected from the group consisting of alcoholic fatty liver, non-alcoholic fatty liver, nutritive fatty liver, starvation fatty liver, obese fatty liver and diabetic fatty liver. A health functional food composition for preventing or improving fatty liver or metabolic syndrome. The method of claim 1,
The metabolic syndrome, obesity, dyslipidemia, hyperlipidemia, diabetes, and a health functional food composition for preventing or improving fatty liver or metabolic syndrome, characterized in that selected from the group consisting of diabetes and insulin resistance syndrome. A pharmaceutical composition for preventing or treating fatty liver or metabolic syndrome comprising thymol as an active ingredient. 13. The method of claim 12,
The thymol is a pharmaceutical composition for preventing or treating fatty liver or metabolic syndrome, characterized in that it is represented by Formula 1
[Formula 1]

. 14. The method of claim 13,
The thymol is a pharmaceutical composition for preventing or treating fatty liver or metabolic syndrome, characterized in that it is separated from the extract of Curcuma Longae . 15. The method of claim 14,
The turmeric extract is a pharmaceutical composition for preventing or treating fatty liver or metabolic syndrome, characterized in that it is extracted with a solvent selected from the group consisting of water, C1 to C4 lower alcohols and aqueous solutions of lower alcohols. 14. The method of claim 13,
The thymol is a pharmaceutical composition for the prevention or treatment of fatty liver or metabolic syndrome, characterized in that it is separated from the fraction of the turmeric extract. 17. The method of claim 16,
The fraction is a pharmaceutical composition for preventing or treating fatty liver or metabolic syndrome, characterized in that the fraction is fractionated with a solvent selected from the group consisting of water, C1 to C4 lower alcohol, aqueous solution of lower alcohol, chloroform and ethyl acetate. 18. The method of claim 17,
The fraction is a pharmaceutical composition for the prevention or treatment of fatty liver or metabolic syndrome, characterized in that the fraction is further fractionated from the ethyl acetate fraction. 13. The method of claim 12,
The thymol is a fat accumulation factor, sterol regulatory element-binding protein-1c (SREBP-1c), acetyl-CoA carboxylase (acetyl-CoA carboxylase, ACC), fatty acid synthase (fatty acid synthase, FAS), CCAAT-enhancer-binding protein (C/EBP), and proliferator-activated receptor γ (PPARγ) A pharmaceutical composition for preventing or treating fatty liver or metabolic syndrome, characterized in that it inhibits. 13. The method of claim 12,
The thymol is a fatty oxidizing factor AMP - A pharmaceutical composition for the prevention or treatment of fatty liver or metabolic syndrome, characterized in that it increases phosphorylation of AMP-activated protein kinase (AMPK). 13. The method of claim 12,
Wherein the fatty liver is selected from the group consisting of alcoholic fatty liver, non-alcoholic fatty liver, nutrient fatty liver, starvation fatty liver, obese fatty liver and diabetic fatty liver. A pharmaceutical composition for preventing or treating fatty liver or metabolic syndrome. 13. The method of claim 12,
The metabolic syndrome, obesity, dyslipidemia, hyperlipidemia, diabetes, and a pharmaceutical composition for preventing or treating fatty liver or metabolic syndrome, characterized in that selected from the group consisting of insulin resistance syndrome. 1) preparing an extract by extracting the dried turmeric with a solvent;
2) preparing a fraction by adding a fractionation solvent to the extract of step 1); and
3) A method for preparing a health functional food composition for preventing or improving fatty liver or metabolic syndrome, comprising purifying thymol derived from turmeric from the fraction of step 2). 1) preparing an extract by extracting the dried turmeric with a solvent;
2) preparing a fraction by adding a fractionation solvent to the extract of step 1); and
3) A method of preparing a pharmaceutical composition for preventing or treating fatty liver or metabolic syndrome, comprising purifying thymol derived from turmeric from the fraction of step 2).

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