KR20240051910A - 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 disease or metabolic syndrome containing thymol derived from turmeric as an active ingredient, and thymol derived from turmeric of the present invention is used in the HepG2 liver cancer cell line in which fat accumulation is induced by oleic acid. When treated with , it 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 related to fat accumulation. , it was confirmed to increase AMPK, a fatty acid oxidation factor, to suppress fat accumulation and improve 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}

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

Fatty liver disease, also called fatty liver disease, 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 is deposited in liver cells, and that the condition then progresses to steatohepatitis (including liver fibrosis), cirrhosis, and hepatocellular carcinoma. In general, causes of fat deposition in the liver include alcohol consumption, obesity, diabetes, abnormalities in lipid metabolism, drugs (steroids, tetracycline, etc.), Cushing syndrome, poisoning (yellow phosphorus, etc.), and severe nutritional disorders.

The causes of fatty liver disease are largely divided into alcoholic and non-alcoholic causes. Liver disease caused by the former is alcoholic fatty liver disease (also called alcoholic liver disorder), and 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 initial simple fatty liver disease 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 patient's liver cells without excessive alcohol consumption. The prevalence of non-alcoholic fatty liver disease continues to increase in parallel with nutritional overnutrition associated with high-fat and high-carbohydrate intake.

Patients with non-alcoholic fatty liver disease usually have obesity, diabetes, and hyperlipidemia. However, not all obese people develop non-alcoholic fatty liver disease, and various factors may play an important role in the development of non-alcoholic fatty liver disease. In particular, non-alcoholic fatty liver disease is divided into primary and secondary depending on the cause. Primary is caused by hyperlipidemia, diabetes, or obesity, which are characteristics of metabolic syndrome, and secondary is caused by nutritional causes (rapid weight loss, starvation, intestinal problems). 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 disease, AIDS infection, HIV infection).

In addition, the accumulation of neutral fat in the liver, which is directly related to non-alcoholic fatty liver disease, and the resulting hepatocellular damage are caused by lipid influx/synthesis into the liver due to changes in local factors and systemic factors such as insulin resistance. It is known that it can occur due to an imbalance in release/oxidation. That is, it is known that hyperinsulinemia caused by insulin resistance causes fatty acids to flow into the liver in excess of the fatty acids that can be oxidized by the mitochondria of hepatocytes, causing neutral fat to accumulate in the liver. Insulin resistance increases the expression of lipogenic transcription factors PPAR-γ (peroxisome proliferator-activated receptor-gamma) and SREBP-1c (sterol regulatory element binding protein-1c), leading to liver lipogenesis. Accumulation of neutral fat may occur due to increased synthesis (de novo hepatic lipogenesis).

Meanwhile, the global incidence of non-alcoholic fatty liver disease is 9-37%, and the prevalence in patients without obesity and diabetes in the Asia-Pacific region is 15-21% and is gradually increasing.

Accordingly, despite the beneficial and therapeutic effects of insulin sensitizers and antioxidants such as metformin and thiazolidinedione, body control through lifestyle changes remains the basis for treatment of non-alcoholic fatty liver disease. It is recognized as efficient and effective. Therefore, much attention is being paid to natural products that effectively increase fat oxidation, reduce lipid production, and regulate lipid metabolism.

Metabolic syndrome is characterized by high levels of blood fat, high blood pressure, insulin resistance, and central obesity (excessive fatty tissue in the abdominal area), and is the basis and cause of all adult diseases, namely obesity, hypertension, hyperlipidemia, and hyperglycemia. This happens. Metabolic syndrome is a phenomenon in which dangerous adult diseases such as arteriosclerosis, high blood pressure, obesity, diabetes, and hyperlipidemia appear simultaneously in one person. Patients with metabolic syndrome live with not only chronic diseases such as diabetes and high blood pressure, but also the risk of sudden death due to cardiovascular disease. Research on metabolic syndrome such as obesity, arteriosclerosis, diabetes, and hyperlipidemia is continuously being conducted.

Turmeric ( Curcuma Longae ) is a perennial plant classified in the genus Curcuma of the Zingiberaceae family. It is native to tropical Asia, is cultivated in India, China, and Southeast Asia, and is used for food, medicine, and as a natural dye. Recently, various papers and media have reported 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.

The purpose of the present invention is to provide a health functional food composition for preventing or improving fatty liver disease or metabolic syndrome containing thymol as an active ingredient.

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

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

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

3) To provide a method for producing a health functional food composition for preventing or improving fatty liver disease 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 1) preparing an extract by extracting dried turmeric with a solvent;

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

3) To provide a method for producing a pharmaceutical composition for preventing or treating fatty liver disease 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 disease or metabolic syndrome containing thymol as an active ingredient.

Additionally, the present invention provides a pharmaceutical composition for preventing or treating fatty liver disease or metabolic syndrome containing thymol as an active ingredient.

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

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

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

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

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

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

The thymol derived from turmeric of the present invention, when treated with the HepG2 liver cancer cell line in which fat accumulation is induced with oleic acid, reduces fat accumulation without cytotoxicity, reduces triglycerides and total cholesterol, and reduces SREBP related to fat accumulation. -1c, it was confirmed that it effectively suppressed the expression of genes and proteins of ACC, FAS, C/EBP and PPAR and increased AMPK, a fatty acid oxidation factor, suppressing fat accumulation and improving fatty liver disease 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 obtained from the 80% methanol extract of turmeric, and Figure 1b is the HPLC of the 50% fermented ethanol extract of turmeric. This diagram shows the results.
Figure 2 is a diagram confirming the cell viability by treating HepG2 cell line with thymol (THY) derived from turmeric of the present invention.
Figure 3 is a diagram showing the inhibition of fat accumulation by treating the HepG2 cell line in which fat accumulation was induced with oleate (OA) with thymol (THY) derived from turmeric of the present invention, and confirming the inhibition of fat accumulation by Oil Red O staining (a: control group, b: group treated with oleic acid alone, c: group treated with turmeric extract, d: positive control group, e: group treated with thymol 100 μM, f: group treated with thymol 200 μM).
Figure 4 shows the treatment of thymol (THY) derived from turmeric of the present invention to the HepG2 cell line in which fat accumulation is induced by oleate (OA), resulting in triacylglycerol (triglyceride, TG) and total cholesterol (total cholesterol). TC) This is the province that confirmed the suppression.
Figure 5 shows the results of quantitative-real-time RT-PCR analysis of the expression of genes related to fat accumulation by treating the HepG2 cell line in which fat accumulation was induced with oleate (OA) with thymol (THY) derived from turmeric of the present invention. (a: SRERP-1c expression confirmed, b: FAS expression confirmed, c: ACC expression confirmed, d: C/EBP expression confirmed, e: PPARγ expression confirmed).
Figure 6 shows the results of Western blot analysis of the expression of proteins related to fat accumulation by treating the HepG2 cell line in which fat accumulation was induced with oleate (OA) with thymol (THY) derived from turmeric of the present invention.
Figure 7 shows the treatment of AMP-activated protein kinase (AMPK), a fat oxidation factor, by treating the HepG2 cell line in which fat accumulation is induced with oleate (OA) with thymol (THY) derived from turmeric of the present invention. This is the result of analyzing phosphorylation by Western blot.

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

The term “prevention” used in the present invention refers to all actions that suppress the symptoms or delay the progression of a specific disease by administering the composition of the present invention.

As used herein, the term “improvement” refers to any action that results in at least a reduction in the severity of a parameter, such as a symptom, associated with the condition being treated.

“Fatty liver” or “fatty liver disease” of the present invention is an indication in which excessive fat (neutral fat) accumulates in the liver. Fatty liver is diagnosed when fat accumulates in more than 5% of the liver weight, and the cause of the disease is mainly caused by excessive drinking. It is divided into alcoholic fatty liver disease and non-alcoholic fatty liver disease caused by factors such as hyperlipidemia, diabetes, and obesity.

According to one embodiment of the present invention, the fatty liver may be selected from the group consisting of alcoholic fatty liver, non-alcoholic fatty liver, nutritional fatty liver, starvation fatty liver, non-alcoholic fatty liver, and diabetic fatty liver. Preferably, non-alcoholic fatty liver. Fatty liver disease, 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, and is 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 typical food composition.

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The above-described flavoring agents include natural flavoring agents (thaumatin), stevia extracts (e.g. rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention can be formulated in the same way 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, gum, candy, ice cream, alcoholic beverages, vitamin complexes, health supplements, etc. There is.

In addition, the food composition contains, in addition to the extract as an active ingredient, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, It may contain alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages.

The functional food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing or treating fatty liver disease or metabolic syndrome. In the present invention, 'health functional food composition' refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with Act No. 6727 on Health Functional Food, and refers to food that is related to the structure and function of the human body. It means taking it for the purpose of controlling nutrients or obtaining useful health effects such as physiological effects. The health functional food of the present invention may contain common food additives, and its suitability as a food additive is determined in accordance with the general provisions and general test methods of the food additive code approved by the Food and Drug Administration, unless otherwise specified. Judgment is made according to specifications and standards. Items listed in the 'Food Additive Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as dark pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum; Examples include mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline preparations, preservative preparations, and tar coloring preparations. For example, the health functional food in the form of a tablet is made by granulating a mixture of the active ingredient of the present invention with excipients, binders, disintegrants and other additives in a conventional manner, and then adding a lubricant and compression molding, or The mixture can be directly compression molded. Additionally, the health functional food in the form of tablets may contain flavoring agents, etc., if necessary. Among capsule-type health functional foods, hard capsules can be manufactured by filling a regular hard capsule with a mixture of the active ingredient of the present invention with additives such as excipients, and soft capsules can be prepared by mixing the active ingredient of the present invention with additives such as excipients. It can be manufactured by filling the mixture with a capsule base such as gelatin. The soft capsule may contain plasticizers such as glycerin or sorbitol, colorants, preservatives, etc., if necessary. The health functional food in the form of a pill can be prepared by molding a mixture of the active ingredient of the present invention and excipients, binders, disintegrants, etc., using a known method. If necessary, it can be coated with white sugar or other coating agent. Alternatively, the surface can be coated with substances such as starch or talc. Health functional food in the form of granules can be manufactured into granules by mixing a mixture of excipients, binders, disintegrants, etc. of the active ingredients of the present invention by a known method, and may contain flavoring agents, flavoring agents, etc., if necessary. You can.

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

According to one 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 lower alcohol aqueous solutions, preferably methanol or Ethanol, but not limited thereto.

According to one embodiment of the present invention, the thymol may be separated from a 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, lower alcohol aqueous solution, chloroform, and ethyl acetate. It may be fractionated with a solvent, preferably an ethyl acetate fraction, but is not limited thereto.

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

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

Additionally, the present invention provides a pharmaceutical composition for preventing or treating fatty liver disease or metabolic syndrome containing thymol as an active ingredient.

The term “treatment” used in the present invention refers to any action that improves or beneficially changes the symptoms of a specific disease by administering the composition of the present invention.

The pharmaceutical composition of the present invention may further include adjuvants in addition to the active ingredients. The auxiliary agent may be any one known in the art without limitation, but the effect may be increased by further including, for example, Freund's complete auxiliary agent or incomplete auxiliary agent.

The pharmaceutical composition according to the present invention can be prepared by incorporating the active ingredient into a pharmaceutically acceptable carrier. Here, pharmaceutically acceptable carriers include 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, Examples include 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 can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions according to conventional methods. .

When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations contain the active ingredient plus at least one excipient, such as starch, calcium carbonate, sucrose, lactose, and gelatin. It can be prepared by mixing etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used diluents such as water and liquid paraffin, they contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. You can. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.

The pharmaceutical composition according to the present invention can be administered to an individual through various routes. All modes of administration are contemplated, for example, by oral, intravenous, intramuscular, subcutaneous, or intraperitoneal injection.

The dosage of the pharmaceutical composition according to the present invention is selected taking into account the age, weight, gender, physical condition, etc. of the individual. It is obvious that the concentration of the active ingredient included in the pharmaceutical composition can be selected in various ways depending on the target, 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 ㎍/ml, pharmaceutical activity may not appear, and if it exceeds 5,000 ㎍/ml, it may be toxic to the human body.

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

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

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

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

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

3) It provides a method for producing a pharmaceutical composition for preventing or treating fatty liver disease 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 through examples. These examples are merely for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

<Example 1> Isolation of thymol derived from turmeric

<1-1> Extraction method and separation method

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

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

To confirm the yield of thymol from turmeric extract (CE), high-performance liquid chromatography analysis (HPLC) analysis was performed. Specifically, dried and sliced turmeric was homogenized using a ball mill (Retsch MM400, Haan, Germany). The homogenized turmeric powder was filtered through a sieve of 40 mesh, 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 in 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 (4.6250mm, 5m), the column oven was maintained at 35°C, and the mobile phase was solvent A (0.1% [v/v] formic acid aqueous solution) and solvent B (0.1% [v/v] formic acid aqueous solution). %[v/v] acetonitrile containing formic acid) was used. The optimal conditions for HPLC are 70% solvent B for 0 to 3 minutes, 50% solvent B for 3 to 16 minutes, and 30% solvent B for 16 to 20 minutes, and analyzed at a flow rate of 1 ml/min, and then using an automatic sample injector. Using , 10 μL of solvent A was injected. Finally, the yield of thymol (THY) isolated from the 50% fermented ethanol extract was confirmed to be 10.67 mg/g (Figure 1B).

<Example 2> Confirmation of improvement in fatty liver

<2-1> Cell culture

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

<2-2> Cell viability analysis

To evaluate the cytotoxicity of thymol, cell viability analysis 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 (Nunc, A/S, Roskilde, Denmark). After inoculating each well to achieve high density, the cells were cultured for 24 hours. After culturing, each well was treated with 200 μg of turmeric extract (CE), 20 μg of positive control (Silymarin, SM), and 50, 100, 200, and 400 μM of thymol (THY), and oleic acid (oleate, OA) to induce fat accumulation. ) was added at 500 μM and cultured for 18 hours (5% CO ₂ , 37°C). After culture, the existing medium was replaced with medium to which MTS solution (5 mg/ml) was added, and after 20 minutes, the microplate reader ( The absorbance of each well was measured at 490 nm using a microplate reader (BioTek South Korea, Seoul, Korea).

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

<2-3> Fat accumulation analysis

For fat accumulation analysis, the HepG2 cell line cultured in Example 2-1 was inoculated into a 24-well plate at a concentration of 4 x 10^5 cells/ml, and 200 μg of turmeric extract (CE) and a positive control (silymarin, 20 μg of Silymarin (SM) and 100 and 200 μM of thymol (THY) were treated, and 500 μM of oleic acid (OA) was added to induce fat accumulation, and cultured for 24 hours. 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. To observe fat accumulated in cells after washing, cells were stained with Oil Red O (Sigma-Aldrich, St. Louis, MO, USA) stain for 1 hour, residual dye was removed with 60% isopropanol, and each well was optically examined. Observed under a microscope.

As a result, as shown in Figure 3, it was confirmed that treatment with oleic acid induced fat accumulation in the HepG2 cell line, a liver cell line, and that treatment with thymol (THY) derived from turmeric suppressed fat accumulation in a concentration-dependent manner. Confirmed.

<Example 3> Lipid metabolism analysis

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 cells/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), and 100 and 200 μM of thymol (THY). To induce fat accumulation, 500 μM of oleic acid (OA) was treated together for 24 days. cultured for some time. After incubation, the levels of TC and TG were measured according to the TC and TG measurement kit protocol, and after completion of the experiment, the absorbance of each well was measured at 450 nm using a microplate reader.

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

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

In order to confirm the effect of thymol derived from turmeric of the present invention on changes in the expression of fat metabolism genes, the expression of fat metabolism genes was analyzed. Specifically, HepG2 cells were inoculated at a concentration of 4 x 10^5 cells/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), and 100 and 200 μM of thymol (THY). To induce fat accumulation, 500 μM of oleic acid (OA) was treated together for 24 days. cultured for some time. Cultured cells were E.Z.N.A. Total RNA was extracted using the Total RNA Kit (Omega Bio-tek, Inc.; Norcross, GA, USA) according to the manufacturer's protocol. For transcription analysis, reverse transcription was performed using the 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). Genes related to fat accumulation include sterol regulatory element-binding protein-1c (SREBP-1c), acetyl-CoA carboxylase (ACC), and fatty acid synthase. Primers for expression analysis of FAS), CCAAT-enhancer-binding protein (C/EBP), and 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 Figure 5, the expression of genes related to fat accumulation, SREBP-1c, ACC, FAS, C/EBP, and PPARγ, increased when HepG2 cell line was treated with oleic acid, and when treated with thymol from turmeric, it increased in a concentration-dependent manner. 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 confirm whether turmeric-derived thymol inhibits 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 turmeric-derived thymol (THY). To induce fat accumulation 1 hour after treatment, oleate (OA) was treated at a concentration of 500 μM and cultured at 37°C for 24 hours with 95% humidity and 5% CO ₂ supplemented. After incubation, the cells were washed twice with DPBS, and the cells were lysed with cell lysis buffer for 2 hours. The lysed cells were centrifuged at 16,000 g for 5 minutes at 4°C for protein extraction. The obtained proteins were quantified according to the quantitative protocol, and the quantified samples were subjected to SDS gel electrophoresis. The electrophoresed protein was transferred to a polyvinylidenedifluoride membrane at 15V for 45 minutes. Membranes were blocked with 3% BSA for 1 h and incubated with SREBP-1c (1:1000), FAS (1:1000), ACC (1:1000), C/EBP (1:1000), PPAR (1:1000), and Primary antibody against pAMPK (1:1000) was used and incubated at 4°C for 18 hours. 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, and 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), a major regulator of lipogenesis and fatty acid oxidation in metabolic tissues, was further confirmed using the Western blot method above, and AMPK (1 : 1000) was used as the primary antibody, and the secondary antibody (1 : 5000) was used.

Primary antibodies against 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 Primary antibodies were purchased from Cell Signaling (Danvers, MA, USA), and antibodies derived from mouse or rabbit were used. The secondary antibody used was goat anti-mouse IgG or goat anti-rabbit IgG.

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

Additionally, as shown in Figure 7, as a result of confirming the phosphorylation of AMPK, it was confirmed that the phosphorylation of AMPK increased when treated with thymol (THY) derived from turmeric, compared to HepG2 cells in which fat accumulation was induced with oleic acid.

<Example 6> Statistical analysis

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

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

Claims (7)

A health functional food composition for preventing or improving nonalcoholic fatty liver disease containing 50% ethanol extract of Curcuma Longae and thymol derived from 80% methanol extract of Turmeric as active ingredients,
The 50% ethanol extract of turmeric contains 10.67 mg/g of thymol,
The thymol derived from the 80% methanol extract of turmeric is thymol isolated from the ethyl acetate fraction of the 80% methanol extract of turmeric,
The thymol inhibits fat accumulation in liver cells and inhibits the production of total cholesterol and triacylglycerol,
The thymol is related to fat accumulation-related SREBP-1c (sterol regulatory element-binding protein-1c), ACC (acetyl-CoA carboxylase), FAS (fatty acid synthase), C/EBP (CCAAT-enhancer-binding protein), and PPARγ ( A composition that inhibits the expression of the gene or protein of proliferator-activated receptor γ) and increases phosphorylation of AMPK (AMP-activated protein kinase, AMPK), a fatty acid oxidation regulator. According to clause 1,
The thymol is a composition represented by Formula 1
[Formula 1]
A pharmaceutical composition for the prevention or treatment of Nonalcoholic Fatty Liver Disease containing 50% ethanol extract of Curcuma Longae and thymol derived from 80% methanol extract of Turmeric as active ingredients,
The 50% ethanol extract of turmeric contains 10.67 mg/g of thymol,
The thymol derived from the 80% methanol extract of turmeric is thymol isolated from the ethyl acetate fraction of the 80% methanol extract of turmeric,
The thymol inhibits fat accumulation in liver cells and inhibits the production of total cholesterol and triacylglycerol,
The thymol is related to fat accumulation-related SREBP-1c (sterol regulatory element-binding protein-1c), ACC (acetyl-CoA carboxylase), FAS (fatty acid synthase), C/EBP (CCAAT-enhancer-binding protein), and PPARγ ( A composition that inhibits the expression of the gene or protein of proliferator-activated receptor γ) and increases phosphorylation of AMPK (AMP-activated protein kinase, AMPK), a fatty acid oxidation regulator. According to clause 3,
The thymol is a composition represented by Formula 1
[Formula 1]
1) Preparing an extract by extracting dried turmeric with 80% methanol;
2) preparing a fraction by adding ethyl acetate to the extract of step 1); and
3) A method for producing a health functional food composition for preventing or improving nonalcoholic fatty liver disease, comprising the step of purifying thymol derived from turmeric from the fraction in step 2),
The thymol inhibits fat accumulation in liver cells and inhibits the production of total cholesterol and triacylglycerol,
The thymol is related to fat accumulation-related SREBP-1c (sterol regulatory element-binding protein-1c), ACC (acetyl-CoA carboxylase), FAS (fatty acid synthase), C/EBP (CCAAT-enhancer-binding protein), and PPARγ ( A method of suppressing the expression of the gene or protein of proliferator-activated receptor γ) and increasing the phosphorylation of AMPK (AMP-activated protein kinase, AMPK), a fatty acid oxidation regulator. 1) Preparing an extract by extracting dried turmeric with 80% methanol;
2) preparing a fraction by adding ethyl acetate to the extract of step 1); and
3) A method for producing a pharmaceutical composition for preventing or treating nonalcoholic fatty liver disease, comprising the step of purifying thymol derived from turmeric from the fraction in step 2),
The thymol inhibits fat accumulation in liver cells and inhibits the production of total cholesterol and triacylglycerol,
The thymol is related to fat accumulation-related SREBP-1c (sterol regulatory element-binding protein-1c), ACC (acetyl-CoA carboxylase), FAS (fatty acid synthase), C/EBP (CCAAT-enhancer-binding protein), and PPARγ ( A method of suppressing the expression of the gene or protein of proliferator-activated receptor γ) and increasing the phosphorylation of AMPK (AMP-activated protein kinase, AMPK), a fatty acid oxidation regulator. In vitro, treating hepatocytes in which fatty acid accumulation is induced with thymol derived from a 50% ethanol extract of Curcuma Longae and an 80% methanol extract of Curcuma longae; Inhibiting fat accumulation in hepatocytes, including; of SREBP-1c (sterol regulatory element-binding protein-1c), ACC (acetyl-CoA carboxylase), FAS (fatty acid synthase), C/EBP (CCAAT-enhancer-binding protein), and PPARγ (proliferator-activated receptor γ). Inhibit gene or protein expression; As a method of increasing phosphorylation of AMPK (AMP-activated protein kinase, AMPK),
The 50% ethanol extract of turmeric contains 10.67 mg/g of thymol,
The method wherein the thymol derived from the 80% methanol extract of turmeric is thymol separated from the ethyl acetate fraction of the 80% methanol extract of turmeric.