KR20190100063A - Composition for preventing, treating, or improving fatty liver comprising of azelaic acid as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing, treating, or alleviating fatty liver containing azelaic acid as an active component, wherein azelaic acid is a compound mainly contained in natural products, thereby having no or little side effects and confirming the efficacy of reducing the accumulation of triglycerides in liver tissues. Therefore, azelaic acid can be used to treat and alleviate fatty liver, and thus can be used to treat steatohepatitis, by confirming an anti-inflammatory effect of azelaic acid. Accordingly, azelaic acid according to the present invention is expected to be usefully used in food materials, pharmaceutical compositions, and health functional foods for treating fatty liver or steatohepatitis.

Description

Composition for preventing, treating, or improving fatty liver comprising of azelaic acid as an active ingredient

The present invention relates to a composition for the prevention, treatment or improvement of fatty liver comprising azelaic acid as an active ingredient.

Fatty liver refers to a condition in which neutral fat accumulates abnormally in liver cells, causing liver enlargement. The main causes of fatty liver include excessive drinking, fatty liver, diabetes, hyperlipidemia, and fatty liver progression can lead to hepatitis, cirrhosis and coronary arteriosclerosis, and can lead to cardiovascular diseases such as myocardial infarction.

Fatty liver includes alcoholic fatty liver and non-alcoholic fatty liver caused by excessive drinking. In general, alcoholic fatty liver can be restored by abstaining from drinking for several months. However, it is important to control carbohydrate intake in non-alcoholic fatty liver. It is not easy to reduce carbohydrate intake because Koreans consume rice as a staple food. The dietary control and natural extracts derived from foods that promote lipolysis in the liver in the form of a pharmaceutical composition or dietary supplements may be effective in improving fatty liver.

Olfr544 (olfactory receptor 544) is a type of G-protein coupled receptors (GPCRs) that are primarily expressed in adipose and liver tissues.In addition to delivering odor information from the olfactory epithelial cells to the cerebrum, the olfactory receptors 544 It is a representative example that is expressed and performs various functions. Recent studies of olfactory receptors expressed in normal tissues have shown that the mouse olfactory receptor family 174 (MOR174) is involved in motility and chemotaxis in sperm and the mouse olfactory receptor family 23 (MOR23) is found in muscle cells of mice. It has been shown to mediate myocyte regeneration and migration. Olfr78 (olfactory receptor 78), an olfactory receptor expressed in the kidneys of mice, regulates the secretion of renin and blood pressure in response to short-chain fatty acids, according to Pluznick et al. This suggests that the olfactory receptors have more functions than previously known.

Azelaic acid (Aza) is an organic compound that contains two carboxyl groups in one molecule, mainly in grains such as wheat, oats, barley, sorghum and rye, and foods such as cranberries. As far as is known, azelaic acid has therapeutic effects on inflammatory skin diseases such as acne and redness, and some studies on atherosclerosis and anticancer activity have been conducted, but the detailed mechanism has not been revealed.

Under these technical backgrounds, the present inventors have completed the present invention by confirming that azelaic acid acts as a ligand of Olfr544 in the liver and mediates PKA-CREB signaling pathway, thereby promoting neutral lipid degradation and improving fatty liver.

Korean Patent Publication No. 10-20150028958

An object of the present invention is to provide a pharmaceutical composition for preventing or treating fatty liver and a food composition for preventing or improving fatty liver including azelaic acid as an active ingredient.

It is an object of the present invention to provide a method of preventing, improving or treating fatty liver by administering an effective amount of azelaic acid to a subject.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to solve the above technical problem, the present invention provides a pharmaceutical composition for preventing or treating fatty liver comprising azelaic acid as an active ingredient, and a method for preventing or treating fatty liver by administering the composition to an individual.

In addition, the present invention provides a food composition for preventing or improving fatty liver comprising azelaic acid as an active ingredient, and a method for preventing or improving fatty liver by administering the composition to an individual.

In one embodiment of the invention, the composition or method may be to activate Olfr544 in liver tissue.

In another embodiment of the invention, the composition or method may be to activate PPARα in liver tissue.

In another embodiment of the invention, the composition or method may be to inhibit triglyceride accumulation in liver tissue.

In another embodiment of the present invention, the composition or method may be to inhibit the accumulation of triglycerides in liver tissue by activating Olfr544 to sequentially activate cAMP-PKA-CREB-PPARα.

In another embodiment of the invention, the fatty liver may be non-alcoholic fatty liver.

In another embodiment of the present invention, the fatty liver may be fatty hepatitis inflamed along with fat deposition on hepatocytes.

The present invention also provides a method for preventing or treating fatty liver, comprising administering azelaic acid to a subject.

The present invention also provides the use of azelaic acid for the manufacture of a medicament for the prevention or treatment of fatty liver.

Azelaic acid according to the present invention is a compound mainly contained in natural products such as cereals and cranberries, such as wheat, oats, barley, sorghum and rye, and has the advantage of having little or no side effects and reducing the accumulation of triglycerides in liver tissue. As confirmed, it can be used for the treatment and improvement of non-alcoholic fatty liver, and also confirmed the anti-inflammatory effect of azelaic acid in liver-derived cell line, it can be used for the treatment of fatty hepatitis. Thus, azelaic acid according to the present invention can be usefully used in food materials, pharmaceutical compositions and health functional foods for the treatment of fatty liver or fatty hepatitis.

1 is a diagram evaluating the cytotoxicity of azelaic acid using the MTT assay.
Figures 2a to 2c is a view showing the cAMP-PKA-CREB mechanism activity according to azelaic acid treatment in Hepa1c1c-7 cells. Specifically, Figure 2a is an increase in cAMP and PKA activity, Figure 2b is an increase in p-CREB protein expression, Figure 2c is a diagram confirming the Olfr544 knockdown effect and the p-CREB protein expression change of the azelaic acid treatment group according to Olfr544 shRNA treatment.
3 is a diagram confirming the increased PPARα gene expression (A), fat oxidation rate (B), and increased PPARα activity (C) through PPRE luciferaseassa in Hepa1c1c-7 cells treated with azelaic acid.
4A to 4E are diagrams confirming the effects of fatty liver inhibition according to azelaic acid treatment in fatty liver induced mice. Specifically, FIG. 4a shows the weight gain inhibition of oral administration of azelaic acid for 6 weeks in normal mice (WT) and Olfr544 KO mice, FIG. 4b is a decrease in triglycerides in liver tissue, and FIG. 4c is an increase in PPARα subgene expression in the liver. 4d is an increase in fat oxidation rate, Figure 4e is a diagram confirming the change in energy metabolism through an indirect calorimetric assay. Figure 4f is a comparison of blood glycerol concentration, body fat amount, insulin sensitivity after long-term administration of azelaic acid in HFD, WT and KO mice.
Figures 5a to 5c is a view confirming the fatty liver inhibition effect by azelaic acid treatment in ob / ob mice. Specifically, FIG. 5A is a view showing weight gain inhibition by oral administration of azelaic acid for 6 weeks in ob / ob mice, FIG. 5B is a decrease in triglycerides in liver tissue, and FIG. 5C is an increase in PPARα subgene expression in the liver. In addition, Figure 5d is a diagram confirming the hepatic lipolytic effect of azelaic acid in mouse primary hepatocyte.
Figure 6 is a comparison of body weight, blood sugar, blood glycerol concentration, cholesterol concentration in rats after the addition of HFD azelaic acid and barley diet.
7A to 7C are diagrams confirming that the inflammatory response is suppressed when azelaic acid is treated in Hepa1c1c-7 induced by inflammation with LPS. Specifically, FIG. 7A shows the cAMP / PKA pathway, calcium / PKC pathway, ERK and JNK signaling pathway, I-3 Kinase / AKT pathway, MEF2 pathway, Hedgehog pathway, and NF-kB pathway in hepa1c1c-7 cells promoted by LPS. , GPCR-dependent transcription factor activity of the JAK / STAT pathway was confirmed, Figures 7b and 7c are IL-6, TNFα mRNA gene expression analysis, C: IL-6, TNFα cytokine concentration analysis.
8a and 8b confirm that the inflammatory response is suppressed when azelaic acid is treated to 3T3-L1 induced inflammation by LPS. Specifically, FIG. 8A illustrates the cAMP / PKA pathway, calcium / PKC pathway, ERK and JNK signaling, I-3 Kinase / AKT pathway, MEF2 pathway, Hedgehog pathway, and NF-kB pathway of LPS-promoted 3T3-L1 cells. , GPCR-dependent transcription factor activity of the JAK / STAT pathway was confirmed, Figure 8b is a diagram analyzing the expression of IL-6, TNFα mRNA genes.
9 is a diagram confirming the change in the gene expression amount after injecting azelaic acid for 3 days to C57BL / 6J male mice after LPS injection. Specifically. A is the gene expression level of an inflammatory marker. B is a gene expression level of growth factors, C and D is a graph of the gene expression level analysis of hepatokine.

The present inventors confirmed that azelaic acid, which is mainly contained in natural products such as wheat, oats, barley, sorghum and rye, and natural products such as cranberries, inhibited the accumulation of triglycerides in liver tissue-derived cell lines. Olfactory receptor 544-cAMP-PKA-cAMP response element binding protein-CPARB-peroxisome proliferator-activated receptor α acts as a ligand for Olfr544, a protein-coupled receptor (GPCR). As a mechanism for activating the pathway of), it was confirmed that it inhibits the accumulation of triglycerides in liver tissue and completed the present invention.

Accordingly, the present invention provides a composition for preventing or treating fatty liver comprising azelaic acid as an active ingredient, and a method for preventing or treating fatty liver by administering the composition to an individual.

In the present invention, "azelaic acid (Azelaic acid, AzA)" is also referred to as nonanediic acid (nonaneedioic acid), has a molecular weight of 188.22 g / mol and a molecular formula of C ₉ H ₁₆ O ₄ has the structure of formula (1).

[Formula 1]

The azelaic acid is a compound mainly contained in natural products such as cereals and cranberries such as wheat, oats, barley, sorghum, rye, and when administered to a subject, has no or little side effects. Studies have shown that the concentration of azelaic acid present in the human body is safe when ingested at about 10-50 μM. In one embodiment of the present invention, as a result of evaluating the cytotoxicity of azelaic acid through the MTT assay, even when treated with a high concentration of azelaic acid was observed that little change in cell viability was confirmed again the safety (See Example 1).

Since azelaic acid of the present invention is a natural substance, it is not toxic and can be continuously used in large amounts as an active ingredient of food or medicine.

The azelaic acid of the present invention can be obtained by conventional extraction methods such as juice extraction, steam extraction, hot water extraction, ultrasonic extraction, solvent extraction, or reflux cooling extraction from natural materials such as cereals such as wheat, oats, barley, sorghum, rye and cranberries. May be selected from the group consisting of water, alcohol having 1 to 4 carbon atoms, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and a mixed solvent thereof. More than one solvent may be used, but is not limited thereto.

In addition, the azelaic acid of the present invention may be a synthetic compound, but it is obvious that it has the same efficacy as that obtained from natural products and can be used for the same use therefrom.

In the present invention, azelaic acid may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful.

As used herein, the term "salt" is useful for acid addition salts formed with pharmaceutically acceptable free acids. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. Obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically nontoxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, and iodide. Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suverate , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, meth Oxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesul Nate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1- Sulfonates, naphthalene-2-sulfonates or mandelate.

The acid addition salts according to the present invention can be dissolved in conventional methods, for example, by dissolving a compound represented by the formula (1) in an excess of an aqueous solution of an acid, which salt is a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. It can be prepared by precipitation using. It may also be prepared by evaporating the solvent or excess acid from the mixture and drying or by suction filtration of the precipitated salt.

Bases can also be used to make pharmaceutically acceptable metal salts. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. The corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg silver nitrate).

In addition, the compounds of the present invention include all salts, isomers, hydrates and solvates that can be prepared by conventional methods, as well as pharmaceutically acceptable salts.

In addition, the composition of the present invention may further include one or more substances commonly used for the prevention or treatment of fatty liver, including azelaic acid as an active ingredient, antihistamines, anti-inflammatory analgesics, anticancer agents, and / or antibiotics, etc. It can be formulated or used in combination with a medicament.

In the present invention, "fatty liver" refers to excessive accumulation of fat in liver tissue due to excessive intake of fat or endogenous increase in liver fat synthesis or decrease in excretion. In general, when fat accumulates in the liver and causes cell damage, it is called fatty liver, but is not limited thereto.

Fatty liver is classified into alcoholic fatty liver and non-alcoholic fatty liver, and alcoholic fatty liver is caused by decreased alcohol metabolism efficiency in liver by excessive drinking, and is known to be prevented, treated or improved by stopping alcohol consumption. Non-alcoholic fatty liver (NAFL) refers to a disease in which triglycerides accumulate in the liver regardless of drinking.

Therefore, the fatty liver disease to be prevented, treated, or improved in the present invention is not limited as long as fat is excessively accumulated in hepatocytes, but preferably means non-alcoholic fatty liver. In a specific embodiment of the present invention, the present inventors treated with azelaic acid in Hepalclc 7 cells, a mouse liver tissue-derived cell line, increased intracellular cAMP and calcium concentrations, increased Olfr544 activity and PKA activity, and increased p-CREB / By confirming the increase in the CREB ratio, it can be seen that azelaic acid acts as a ligand to Olfr544 in hepatocytes to activate the cAMP-PKA-CREB signaling mechanism (see Example 2).

In addition, in a specific embodiment of the present invention, as a result of treatment with azelaic acid on Hepalclc 7 cells, it was confirmed that the expression and activity of PPARα were increased and the fat oxidation rate was increased compared to the control group (see Example 3). As a result of analyzing oral administration of 50 mg / kg of azelaic acid, the concentration of triglycerides in liver tissues was found to be significantly decreased in the liver tissues. It was confirmed that administration reduced non-alcoholic fatty liver symptoms by promoting fatty oxidation in mice, and compared blood glycerol concentration, body fat mass and insulin sensitivity after prolonged administration of azelaic acid in HFD, WT and KO mice (see Example 4). ).

In addition, in a specific embodiment of the present invention, after six weeks of oral administration of azelaic acid in ob / ob mice, an obese mouse model, hepatic neutral lipids, PPARα, a fatty acid regulation regulator, and azelaic acid in an obese model through analysis of subgene expression. The non-alcoholic fatty liver improvement effect of was re-verified, and hepatic lipolytic effect of azelaic acid was reconfirmed in mouse primary hepatocytes (see Example 5).

In addition, the weight, blood glucose, blood glycerol concentration, and cholesterol concentrations of rats after diet with HFD azelaic acid and barley were compared. The weight, LDL cholesterol concentration, ALT concentration, and liver in azelaic diet group compared to barley diet group. As a result, it was found that a significantly reduced effect on tissue triglyceride concentration showed that azelaic acid showed superior efficacy compared to barley diet, which is generally effective in alleviating fatty liver (see Example 6). .

That is, in the present invention, the treatment of fatty liver may be characterized by a decrease in neutral lipids in blood and liver tissues and inhibition of lipid accumulation in liver tissues, and azelaic acid targets olfr544, an olfactory receptor expressed in liver tissues, especially liver tissues. It acts as an active action of PPARα, may be to promote fatty acid oxidation.

On the other hand, non-alcoholic fatty liver includes simple fatty liver (steatosis) and non-alcoholic steatohepatitis (NASS). Simple fatty liver is considered to be a benign disease with a good prognosis, but nonalcoholic steatohepatitis is a progressive liver disease that causes cirrhosis and liver cancer.

In a specific embodiment of the present invention, the inventors have confirmed the anti-inflammatory activity of azelaic acid in addition to the inhibitory effect of the accumulation of azelaic acid in the liver tissues, more specifically inflammatory cytokines according to the treatment of azelaic acid in liver tissue-derived cell line It was confirmed that the expression of the kines IL-6 and TNF-α can be reduced to effectively reduce inflammation by treating azelaic acid in hepatocytes (see Examples 7 to 9). The composition of the present invention can be used as a composition for the prevention or treatment of hepatitis in addition to the treatment of fatty liver, in particular to be used as a composition for the prevention or treatment of fatty liver disease accompanied by inflammation of fatty liver by fat accumulation in liver tissue. Can be.

In the present invention, "prevention" means any action that inhibits fat accumulation or delays the development of fatty liver in liver tissue by administration of the composition according to the present invention, and "treatment" means fatty liver by administration of the composition according to the present invention. By "administrative" is meant any action that improves or advantageously alters the symptoms of, and reduces the parameters associated with fatty liver disease, such as the extent of symptoms by administration of a composition according to the present invention.

In the present invention, the fatty liver improvement may be characterized by inhibition of degradation and accumulation of neutral lipids in liver tissue and thus improvement of vascular health.

In the present invention, the pharmaceutical composition may further include appropriate carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions.

As used herein, the term "carrier" refers to a compound that facilitates the addition of a compound into a cell or tissue. For example, dimethyl sulfoxide (DMSO) refers to a cell or It is a commonly used carrier that facilitates the incorporation of many organic compounds into tissues.

As used herein, "diluent" is defined as a compound that not only stabilizes the biologically active form of the compound of interest, but also is diluted in water to dissolve the compound. Salts dissolved in buffer solutions are used as diluents in the art. A commonly used buffer solution is phosphate buffered saline, because it mimics the salt state of human solutions. Because buffer salts can control the pH of a solution at low concentrations, buffer diluents rarely modify the biological activity of a compound. Compounds containing azelaic acid as used herein may be administered to a human patient as such or as a pharmaceutical composition, mixed with other ingredients or with a suitable carrier or excipient, such as in combination therapy.

In addition, the pharmaceutical composition for preventing or treating fatty liver containing azelaic acid according to the present invention is an external preparation such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and sterile injectable solutions according to conventional methods, respectively. Carriers, excipients and diluents that may be used in the form of azelaic acid include, but are not limited to, lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and Mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, or the like. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspending agents, liquid solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Therapeutically effective amounts for the compounds containing azelaic acid of the present invention can be determined initially from cell culture assays. For example, the dose can be calculated in an animal model to obtain a range of circulating concentrations including a half maximal inhibitory concentration (IC50) or a half maximal effective concentration (EC50) determined in cell culture. Such information can be used to more accurately determine useful doses in humans. The dosage of azelaic acid can vary within this range depending upon the dosage form employed and the route of administration utilized.

The subject to which the azelaic acid of the present invention is administered refers to a subject to which a composition containing azelaic acid to azelaic acid as an active ingredient may be administered, and the subject is not limited and includes, for example, a mammal such as a human.

Preferred dosages of the compositions comprising azelaic acid to azelaic acid according to the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. have. However, for the desired effect, the pharmaceutical composition of the present invention is preferably administered at 0.0001 to 1000 mg / kg, preferably 0.5 to 200 mg / kg, more preferably 0.5 to 100 mg / kg per day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The pharmaceutical composition according to the present invention can be administered to mammals such as rats, mice, livestock, humans by various routes, such as parenteral, oral, and all modes of administration can be expected, for example, oral, It may be administered by rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

In addition, the oral dosage form may vary depending on the age, sex, and weight of the patient, but the amount of 0.1 to 100 mg / kg may be administered once to several times a day. In addition, the dosage may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

In the present invention, when provided with a mixture in which other components are added in addition to azelaic acid, the composition may contain 0.001% to 99.9% by weight of the azelaic acid, preferably 0.1% to 99.0% by weight, more preferably, based on the total weight of the composition. Preferably from 30% to 50% by weight.

In addition, the present invention provides a food composition for preventing or improving fatty liver, comprising azelaic acid as an active ingredient. In addition, azelaic acid may be added to food for the purpose of improving fatty liver or vascular disease. When the azelaic acid of the present invention is used as a food additive, the azelaic acid can be added as it is or used with other food or food ingredients, and can be suitably 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). In general, the azelaic acid of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less based on the raw material in the manufacture of food or beverage. However, in the case of long-term intake for health and hygiene or for health control, the amount 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.

In the present invention, the food includes a functional food and a health functional food, and the term 'functional food' refers to a food in which the functionality of the general food is improved by adding the azelaic acid of the present invention to the general food. Functionality can be roughly divided into physical and physiological functions. When the azelaic acid of the present invention is added to a general food, the physical and physiological properties of the general food will be improved. It is broadly defined as 'functional food'.

Functional foods of the present invention can be used in a variety of drugs, foods and beverages for the prevention or improvement of alcoholic and / or non-alcoholic fatty liver by reducing the accumulation of liver tissue fat. 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 the like and include all foods in a conventional sense.

The health beverage composition according to the present invention may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in the general beverage. Natural carbohydrates described above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The proportion of the natural carbohydrate is generally about 0.01-20 g, preferably about 5-12 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 usually chosen in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention.

As the invention allows for various changes and numerous embodiments, particular embodiments will be illustrated in the drawings and described in detail in the written description. However, this is not intended to limit the present invention to specific embodiments, it should be understood to include all transformations, equivalents, and substitutes included in the spirit and scope of the present invention. In the following description of the present invention, if it is determined that the detailed description of the related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

EXAMPLE

Example 1 Cytotoxicity Assessment of Azelaic Acid

MTT assay was performed to evaluate the cytotoxicity of azelaic acid. The MTT assay is a test that utilizes the ability of the mitochondria to reduce the yellow water-soluble substrate MTT tetrazolium to a blue-violet water-insoluble MTT formazan by dehydrogenase action. MTT reagent was prepared by diluting at a concentration of 2-5 mg / mL in PBS (Phosphate buffered saline). Hepa1c1c-7 cells used in the experiment were purchased from Korea Cell Line Bank and cultured by adding 10% FBS and 1% PEST to minimum essential medium Eagle alpha modification medium (MEM-alpha, Hyclone). For experiments, dispensing Hepa1c1c-7 cells (4 × 10 ⁴ cells / mL) in a 96-well plate, incubating at 37 ° C., 5% CO _{2 for} 24 hours, and then adding 0 to 500 μM of azelaic acid. The cells were incubated in the concentration section for 24 hours. Then, 100 μL of MTT reagent was added to each sample at a concentration of 4 mg / mL, followed by incubation for 4 hours at 37 ° C. and 5% CO ₂ , and then 100 μL of DMSO (dimethyl sulfoxide) was measured for absorbance at 540 nm. Absorbance values quantify the effect of cell death by toxicity using a principle proportional to the number of viable cells.

Cytotoxicity experiments on 3T3-L1 adipocytes were performed by Tetrazolium-based colorimetric (MTT) method. The MTT assay showed that the dehydrogenase in the mitochondria of intact metabolism was reduced to the yellow water-soluble tetrazoliumsalt [3- (4,5-dimethylthiazol-2-yl) -2-5-diphenyltetrazolium bromide] (MTT), insoluble dark purple MTT. It is based on the principle of reducing tomazan crystals. It is a method of evaluating cytotoxicity by measuring absorbance at an appropriate wavelength (mainly 500 to 600 nm). As a result, as shown in Figure 1, when treated to Hepa1c1c-7 cells and 3T3-L1 adipocytes at a concentration of 0 ~ 500 μM azelaic acid it was confirmed that no toxicity compared to the control at all concentrations.

Example 2 Study of Lower Signaling Mechanisms through Olfr544 Activation by Azelaic Acid Treatment

2-1) Olfr544 knockdown cell line construction

To more clearly confirm that azelaic acid acts as a ligand of Olfr544, Olfr544 knockdown cell lines were constructed using vectors containing shRNA, and the following experiments were performed to compare protein expression levels of normal and knockdown cells. For the construction of Olfr544 knockdown cell lines, specifically, oligonucleotides encoding a shRNA hairpin sequence targeting Olfr544 (top strand [SEQ ID NO: 1]: 5′-CACCGCTCACTGTTCGCATCTTCATTCGAAAATGAAGATGCGA-ACAGTGAG-3 ′) or any shRNA hairpin that does not target Oligonucleotides (top strand [SEQ ID NO: 2]: 5′-CACCGTAAGGCTATGAAGAGATACCGAAGTATCTCTTCATAGCCTTA-3 ′) encoding non-targeting scrambled shRNA hairpins were shRNAs of the pENTR / U6 vector using the Block-iT U6 RNAi Entry Vector Kit (Invitrogen). Inserted into cloning site. Next, Olfr544 knockdown cell lines were prepared by seeding Hepa1c1c-7 or 3T3-L1 cells in a 6-well plate and transfecting 2.5 μg Olfr544 shRNA or scrambled shRNA with 10 μL Lipofectamin2000 (Invitrogen) for 48 hours. The prepared cells were cultured in 5% CO ₂ , 37 ° C., and humidified conditions.

2-2) cAMP concentration measurement

The cAMP assay uses the principle of estimating the amount of cAMP that is a secondary messenger of the ligand-activated GPCR receptor, using the cAMP standard calibration curve. The 96 well-plates included in the cAMP Assay ELISA kit (Enzo Life science, New York, USA) were coated with GxR IgG antibodies, and cAMP in blue solution for standard calibration curves was combined with alkaline phosphatase, It turns yellow by rabbit antibodies. This causes a change in color when the wells bound with cAMP are activated by the addition of pNpp substrate, and the cAMP concentration can be measured according to colorimetric quantification at a wavelength of 405 nm.

In this example, Hepa1c1c-7 cells were differentiated into 1 x 10 ⁴ cells per well in 96-well plates, followed by 50 μM azelaic acid (AzA) and positive control 1 μM Foskolin (FSK) for 18 hours. Treatment was performed to determine cAMP concentrations as described above.

2-3) protein extraction

Hepa1c1c-7 cells (or adipose tissue) are rinsed with phosphate-buffered saline (PBS) to collect only cell (or tissue) pellets, and solution C (120 mM NaCl, 5 mM KCl, 1.6 mM MgSO ₄ , 25 mM). After washing with NaHCO ₃ , 7.5 mM D-glucose, pH 7.4), Solution D (solution C + 10 mM CaCl ₂ ) was added thereto, and the mixture was stirred for 20 minutes and centrifuged. The supernatant obtained after centrifugation was again centrifuged to dissolve the precipitate obtained in TEM buffer solution (10 mM Tris, 3 mM MgCl ₂ , 2 mM EDTA, pH 8.0) and glycerol (membrane protein). The final result was obtained in fractions. The protein extraction method as described above is known as a calcium ion bombardment method with a higher protein yield than the conventional mechanical membrane fractionation method. Meanwhile, when obtaining a cytosol protein fraction, Hepa1c1c-7 cells (or adipose tissue) are rinsed with PBS to collect only pellets and a buffer solution containing digitonin (digitonin) (150 mM NaCl, 50 mM HEPES, 25 μg / mL digitonin, pH 7.4) was added and allowed to stand on ice for 10 minutes, followed by centrifugation to finally obtain supernatant as a cytosol protein fraction. On the other hand, when obtaining a whole protein (whole protein extract) without fractionation, hepa1c1c-7 cells were added to the cell lysis buffer (50 mM Tris, 1% Triton-X 100, 1mM EDTA, PI (proteinase inhibitor)) The cells were lysed and then centrifuged at 4 ° C. for 20 minutes at 13,000 rpm to obtain a supernatant.

2-4) Protein Electrophoresis (SDS-PAGE) and Western Blot

In order to confirm whether the Olfr544 protein is expressed, the protein fractions (membrane protein, cytosol protein) and whole protein extract extracted by the method of Example 2-3 are Bradford. It was quantified by the method. Each sample takes about 40 μg of protein and is heated and / or dissolved appropriately depending on the type of protein extracted (fractionated protein, total protein or phosphorylated protein, etc.) to use 12% SDS- PAGE was performed. Western blot was blocked to prevent nonspecific binding to nitrocellulose membranes (NC membranes) containing proteins blotted from SDS-PAGE gels, and then the primary antibody to the protein to be detected ( Anti-Olfr544 rabbit antibody (Abcam, UK), anti-β-actin antibody (loading control, Santa Cruz, CA, USA) and secondary antibody (HRP-conjugated-anti-rabbit IgG, Santa Cruz, California, USA) were treated sequentially and then each at room temperature for 1 hour. The expression level of each gene was normalized using β-actin or GAPDH expression, and the content of protein bands in the Western blot was quantified and quantified by software (Gel-Pro Analyzer 4.0). The antibodies used in the western blot were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA USA).

2-5) Confirmation of Olfr544 Activation and its Downstream Signaling Pathway by Azelaic Acid

As shown in FIG. 2a, it was confirmed that the cAMP and calcium concentrations in the cells were significantly increased in the Hepa1c1c-7 cells compared with the control group, and that the activity was significantly increased in accordance with the azelaic acid treatment in the PKA activity. I could confirm it.

In addition, as shown in Figure 2b, it was confirmed that the p-CREB / CREB ratio significantly increased by the activity of phosphorylation of CREB in the azelaic acid treatment group.

On the other hand, as can be seen in Figure 2c, the increase in the p-CREB / CREB ratio was not observed in cells subjected to knockdown of Olfr544 by transfection.

Taken together, it can be seen that azelaic acid acts as a ligand to Olfr544 to activate cAMP-PKA-CREB signaling mechanism.

Example 3. Confirmation of the effect of azelaic acid on PPARα activity

3-1) quantitative real-time PCR (qPCR)

In order to confirm the change in the expression level of PPARα gene by azelaic acid, Hepa1c1c-7 cells were cultured in a 24-well plate at a concentration of 5 × 10 ⁵ cells / mL and incubated for 24 hours. Treated for hours. RNA was extracted and cDNA synthesis was performed using the extracted RNA using the ReverTra Ace® qPCR RT kit (TOYOBO, Osaka, Japan). More specifically, in order to increase the efficiency of the PCR reaction, the extracted RNA was treated at 65 ° C. for 5 minutes and immediately stored on ice. Thereafter, a total of 8 μL of a reactant was prepared with 2 μL of 4x DNA Master Mix, 0.5 μg RNA, and distilled water (nuclease-free water) containing a gDNA scavenger and reacted at 37 ° C. for 5 minutes. Subsequently, 5x RT Master mix was added to the reaction product, and cDNA was synthesized by reacting for 37 ° C.-5 min, 50 ° C.-5 min, and 98 ° C.-5 min. CDNA synthesized by the above method was performed by qPCR using THUNDERBIRD SYBR qPCR Mix (TOYOBO) and iQ5 iCycler system (Bio-rad, CA, USA) by methods known to those skilled in the art. After denaturation for 30 seconds, the denaturation process was further performed over 40 cycles for 10 seconds at the same temperature, followed by heating and cooling for 30 seconds at 55 ° C to 60 ° C, and stretching at 68 ° C for 20 seconds. Each gene expression level was normalized using GAPDH expression. Primer designs were designed using nucleotideblast software from the National Center for Biotechnology Information (NCBI) and purchased from Bionics (Seoul, Korea).

3-2) Fatty acid oxidation rate measurement

Hepa1c1c-7 cells were cultured in 24-well plates at a concentration of 5 × 10 ⁵ cells / mL and incubated for 24 hours, and then treated with azelaic acid at 100 μM for 24 hours. After accumulating [1- ¹⁴ C] palmitate and incubating for one hour, the medium was collected and CO ₂ was extracted using NaOH and HCl to measure ¹⁴ CO ₂ with a liquid scintillation counter. GW7647 (1 μM) was used as a positive control.

3-3) PPRE-luciferase assay

For the determination of PPRE-luciferase, HEK293 cells (Korea Cell Line Bank) dispensed at 2 x 10 ⁵ cells per well in 24 well plates. pSG5-PPAR alpha (Addgene, MA, USA) was co-transfected according to the given protocol using pCMV-3xPPRE-Luc, Renilla expression vector and Lipofectamine 2000 (Invitrogen). After 24 hours post-transfection, cells were treated with various concentrations of azelaic acid and analyzed using a dualluciferaseassay kit (Promega, Fitchburg, WI, USA) and Victor X2 (PerkinElmer, Santa Clara, CA). Luciferase fluorescent signals were normalized using Renilla.

3-4) PPARα activation by azelaic acid and its mechanism

As shown in FIG. 3, treatment with azelaic acid in Hepa1c1c-7 cells induced a significant increase in expression of PPARα (A), and fatty acid oxidation was significantly increased compared to the control (B). In the results of PPRE-luciferase, the effect of increasing the fat oxidation through the PPARα activation of azelaic acid confirmed that azelaic acid does not act as a direct ligand to PPARα (C). According to the results, it can be seen that azelaic acid activates Olfr544, thereby activating cAMP-PKA-CREB, thereby activating PPARα and thus increasing the fatty acid oxidation rate.

Example 4. Confirmation of fatty liver inhibition of azelaic acid in fatty liver induced mice

4-1) Experimental animal model creation

The mice used in this example were purchased from Samtako, an 8-week-old C57BL / 6J model, and Olfr544 KO mice were prepared from Macrogen through the CRISPR / Cas9 system based on the C57BL / 6J model. All experiments conducted in this example were conducted according to the protocol passed by Korea University Laboratory Animal Ethics Committee (Protocol No. KUIACUC-20170322-1). Mice were acclimated for 12 hours in an environment of 21-25 ° C and 50-60% relative humidity, and used in the experiments. Agela diluted in distilled water at a concentration of 50 mg / kg of body weight for 6 weeks in a total of 18 mice. Oral administration of acid and distilled water as a control group was performed. Mice used in the experiment induced fatty liver by feeding a high fat diet containing 60% fat (D12492, Research Diets, Inc), and weighed weekly.

4-2) Analysis of neutral lipid concentration in liver tissue

Neutral lipid concentration measurement in liver tissue was carried out by homogenizing the collected liver tissue in 600 μL acetone (Daejeong, Korea) for 24 hours at 4 ℃ and centrifuged at 12000 rpm for 10 minutes to obtain fat parts It was. Fatty parts were dried and dissolved in 95% ethanol and quantified by Cobas C11 autoanalyzer (Roche, Basel, Switzerland) and averaged using liver weight.

4-3) Analysis of PPARα and Submechanism Gene Expression in Liver Tissues

RNA was extracted from liver tissue and the expression of PPARα and PPARα subgenes Acox1, Cpt1a, Hmgcs2 were analyzed by qPCR by the method of Example 3-1).

4-4) Fatty acid oxidation rate measurement

To evaluate fatty acid oxidation, 100 mg of liver tissue was homogenized in a cold sucrose solution at 0.25 M concentration, followed by centrifugation at 600 rpm and 4 ° C to obtain a supernatant, followed by supernatant 450. 50 μL of 10% Triton X-100 was added to μL. 5 μL of the sample was then 950 μL 50 mM Tris-HCl, 10 μL 20 mM NAD, 3 μL 0.33 M Dithothereitol, 5 μL 1.5% BSA, 5 μL 2% Triton X-100, 10 μL 10 mM CoA, and 10 μL Mixed with 1 mM FAD. 2 μL of 5 mM palmitoyl-CoA was added to the mixture at 37 ° C., and the results were analyzed for 5 minutes at 340 nm using a spectrophotometer.

4-5) Indirect calorimetric assay

C57BL / 6J mice and Olfr544 KO mice were treated with metabolic cages for 3 days after HFD and azelaic acid for 6 weeks, and the results of day 3 were analyzed and analyzed. Oxygen consumption (VO ₂ ) and carbon dioxide production (VCO ₂ ) were analyzed using Oxylet Physiocage System (Panlab / Harvard apparatus, Cornella, Spain) and suiteMETABOLISM (V2.2.01, Panlab) software. Respiratory quotient (RQ) was calculated by the VCO ₂ / VO ₂ formula and energy expenditure was analyzed by the formula EE = VO ₂ x 1.11 x (3.815 + 1.232 x RQ). Oxidation of free fatty acids was calculated according to the formula (1.6946 x VO ₂ )-(1.7012 x VCO ₂ ).

4-6) Confirmation of Fatty Liver Inhibitory Effect of Azelaic Acid in Fatty Liver Mice

Through the above experiment, it was confirmed that the body weight was reduced compared to the control group when orally administered azelaic acid for 6 weeks with high fat diet administration (FIG. 4A), and it was confirmed that the triglyceride concentration in the liver tissue was reduced (FIG. 4B). In addition, significantly increased expression of PPARα and its target gene in liver tissue (FIG. 4C), and then measured the fat oxidation rate in liver tissue, and it was confirmed that the fat oxidation rate was increased compared to the control (FIG. 4D). Indirect calorimetric analysis showed decreased respiratory rate and increased free fatty acid oxidation, suggesting that fatty acid oxidation was promoted in mice by azelaic acid, but not in Olfr544 KO mice knocked down Olfr544 (FIG. 4E). . The results suggest that long-term administration of azelaic acid may reduce body weight and lower triglyceride levels in liver tissue, and azelaic acid may increase the expression of PPARα and its target genes in liver tissue and promote fatty acidization. According to the above results, long-term administration of azelaic acid reduces fatty liver, and this effect is found to be dependent on Olfr544.

4-7) Comparison of blood glycerol concentration, body fat mass and insulin sensitivity after long-term administration of azelaic acid in HFD, WT and KO mice

Azelaic acid (100 mg / kg) was administered intraperitoneally and the concentration of glycerol, a marker for body fat breakdown, was measured. In WT mice, azelaic acid promoted body fat breakdown, but it was ineffective in KO mice. Azelaic acid 50 mg / kg / in WT and KO mice. As a result of the day and body fat analysis by microCT method, total fat and abdominal fat decreased significantly in WT mice, but this effect was not observed in KO mice, indicating that azelaic acid promotes body fat by activating Olfr544 ( 4f). To determine glucose tolerance and insulin resistance, glucose and insulin resistance tests were performed in mice administered with glucose (1.5 kg / bw) and insulin (0.35 unit / kg / bw). This effect was not observed in KO mice, but azelaic acid improved glucose tolerance and insulin resistance.

Example 5 Confirmation of Fatty Liver Inhibitory Activity of Azelaic Acid in ob / ob Mice

5-1) Experimental Animal Model

Male 7-week-old fatty liver model mouse ob / ob mice were purchased from a central laboratory animal and used for the experiment. Azelaic acid was orally administered at a concentration of 50 mg / kg of body weight for six weeks at the same time as a 60% high fat diet (HFD) diet. Weight was measured once a week during the experiment, and feed intake was performed three times, twice a week in the last week.

5-2) Analysis of neutral lipid concentration in liver tissue

Neutral lipid concentration measurement in liver tissue was carried out by homogenizing the collected liver tissue in 600 μL acetone (Daejeong, Korea) for 24 hours at 4 ℃ and centrifuged at 12000 rpm for 10 minutes to obtain fat parts did. Fatty parts were dried and dissolved in 95% ethanol and quantified by Cobas C11 autoanalyzer (Roche, Basel, Switzerland) and averaged using liver weight.

5-3) Fatty acid oxidation rate measurement

The fatty acid oxidation rate was measured and analyzed by the method of Example 4-4).

5-4) Confirmation of Fatty Liver Inhibitory Effect of Azelaic Acid in ob / ob Mice

Through the experiment, it was confirmed that weight loss compared to the control group when oral administration of azelaic acid for 6 weeks with high fat diet administration to ob / ob mice (Fig. 5a), it was confirmed that the concentration of triglycerides in liver tissue (Fig. 5b) ). In addition, it was confirmed that significant expression of PPARα and its target genes increased in liver tissue (FIG. 5C). The results suggest that long-term administration of azelaic acid reduces body weight in ob / ob mice, lowers triglyceride levels in liver tissue, and significantly increases the expression of PPARα and its target gene Hmgcs2 gene in liver tissue. According to the above results, it can be seen that azelaic acid can be utilized in various forms by oxidizing the lipids accumulated in the liver to prevent and treat fatty liver.

5-5) Confirmation of Hepatic Lipolytic Effects of Azelaic Acid in Mouse Primary Hepatocytes

To reconfirm the hepatic lipolytic effect of azelaic acid, experiments were carried out using mouse primary hepatocytes. Experimental results showed that azelaic acid significantly increased Ppara, Acox1, and Cpt1a, major gene markers involved in fatty acid degradation in mouse primary hepatocytes, as in Hepa1c1c cells, which are liver tissue cell lines (FIG. 5D). It was reaffirmed that palpation could treat / prevent fatty liver lesions.

Example 6 Comparison of Body Weight, Blood Glucose, Blood Glycerol Concentrations and Cholesterol Concentrations in Rats After Diet with HFD Azelaic Acid and Barley

To reconfirm the inhibitory effect of azelaic acid on the treatment of fatty liver, a comparative experiment was conducted with barley-added diet, which is already known to prevent fatty liver. Mice were fed diets containing azelaic acid (0.5 g / kg diet) and 10% (w / w) barley in high fat diet for 6 weeks. In the high fat and barley diet groups, there was no weight loss, but the weight was significantly decreased in the azelaic acid group. Serum fasting glucose, triglyceride, total cholesterol, and HDL cholesterol were not significantly different among the groups. However, the azelaic acid group showed a significant decrease in LDL cholesterol levels. It was confirmed that there is an effect to protect tissue damage. In addition, the liver tissue triglyceride concentration, which is an indicator of fatty liver lesions, was significantly decreased in the azelaic acid group, and showed superior efficacy compared to barley diet, which is generally effective in alleviating fatty liver (FIG. 6).

Example 7 Azela Inflammation Reduction Effect in Hepa1c1c-7 Cells

7-1) Cell Culture

Mouse hepatocyte Hepa1c1c-7 was purchased from Korea Cell Line Bank (Seoul, Korea). Hepa1c1c-7 cells. 10% FBS and 1% PEST were added using a minimum essential medium Eagle Alpha Modification Medium (MEM-α, Hyclone) under 5% CO ₂ , 37 ° C.

7-2) Induction of inflammation and treatment of azelaic acid by LPS

Hepa cells of mouse Hepa1c1c-7 were inoculated in 96 well-plate at 1X10 ⁴ concentration and cultured for 24 hours, followed by transfection of the construct containing the promoter of transcription factor and luciferase for 8 hours. The experiments were performed in two groups: LPS-induced hepatic inflammation and no LPS treatment. The untreated control group was treated with 50 μM of azelaic acid for 6 hours, and the experimental group induced inflammation with 100 ng / mL of the inducing substance LPS for 1 hour, and then treated with azela 50 μM for 6 hours. The subpath activated by the acid was identified.

7-3) Ten Signaling Path Experiments of GPCR

In order to confirm the mechanism of anti-inflammatory effect of azelaic acid, 10 signal transcribing transcription factors of GPCR were analyzed. Previous patents have confirmed that azelaic acid activates olfactory receptor Olfr544, a GPCR, in mouse adipose progenitor cells 3T3L1, and this mechanism promotes lipolysis through cAMP-CREB signal transduction (Korean Patent 10-15932539). . On the basis of the Olfr544-cAMP-CREB signal transduction axis of azelaic acid, we examined the changes in the complex lower signaling pathway system by GPCR 10 pathway reporter assay using 10 transcription factors. .

The measurement of GPCR's 10 signal transduction pathways is based on the reporter gene assay method, which measures typical signal paths of GPCRs.It uses 10 transcription factors to change the complex sub-signal pathway system according to GPCR activity and inhibition. You can look at it. The GPCR Signaling 10-pathway Reporter Array (QIAGEN, Netherlands) used in this example contains a transcription factor and a firefly luciferaseconstruct that induce sub-signaling in each experimental group, which continuously expresses the Renilla luciferaseconstruct, which activates luciferase activity. This is the principle to measure. The 10 transcription factors are: ATF2 / ATF3 / ATF4, CREB, ELK1 / SRF, FOS / JUN, MEF2, GLI, FOXO, STAT3, NFAT, NFκB. In this Example, luciferase activity was measured using a dualluciferaseassay kit (Promega, USA) and Victor ^TM X2 (PerkinElmer, CA, USA).

7-4) cDNA Synthesis

1 μg of RNA extracted from Hepa1c1c-7 hepatocytes was quantified and cDNA synthesized using ReverTra Ace qPCR RT kit (TOYOBO, Osaka, Japan). More specifically, in order to increase the efficiency of the RT-PCR reaction, the extracted RNA was treated at 65 ° C. for 5 minutes and immediately stored on ice. Thereafter, a total of 8 μL of a reactant prepared with 2 μL of 4x DNA Master Mix, 0.5 μg RNA, and distilled water (nuclease-free water) containing a gDNA remover was reacted at 37 ° C. for 5 minutes. Then, 5x RT Master mix was added to the reaction product, and cDNA was synthesized by reacting for 37 ° C.-5 min, 50 ° C.-5 min, and 98 ° C.-5 min.

7-5) Quantitative real-time PCR (qPCR)

CDNA synthesized by the above method was performed using a THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka, Japan) and an iQ5 iCycler system (Bio-rad, California, USA). More specifically, after denaturation at 95 ℃ for 4 minutes and 30 seconds, after further modification over 40 cycles (cycle) for 10 seconds at the same temperature, and then heated and cooled for 30 seconds at 55 ℃ ~ 60 ℃ Elongation was performed at 68 ° C. for 20 seconds. Each gene expression level was normalized using GAPDH expression. Primer designs were designed using nucleotideblast software from the National Center for Biotechnology Information (NCBI) and were purchased from Bionics (Seoul, Korea).

7-6) Analysis of IL-6 and TNF-α Cytokine Concentrations in Hepa1c1c-7 Cells

Hepa1c1c-7 cells were incubated for 24 hours in 12-well plates. The cells were pretreated with 50 μM of azelaic acid for 1 hour and then treated with 100 ng / mL LPS for 6 hours in the presence of azelaic acid. After lysis in Hepa1c1c-7 cells with lysis buffer (50 mM Tris, 1% Triton-X 100, 1 mM EDTA, proteinase inhibitor), centrifuge at 13,000 rpm, 4 ^o C, 20 minutes to obtain supernatant. Thermo scientific (IL, USA), mouse IL6 ELISA kit and mouse TNFα ELISA kit was analyzed using. Tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) was quantified using ELISA methods according to the instructions of the skilled artisan.

7-7) Confirmation of Azela Inflammation Reduction Effect in Hepa1c1c-7 Cells

7A shows the results of analyzing the transcription factor change of the inflammation-related GPCR-dependent signaling pathway by azelaic acid in mouse hepatocyte Hepa1c1c-7 by the method of Example 7-3. As can be seen in Figure 7a, after the LPS treatment of Hepa11c-7 cells induce inflammation, the activity of transcription factors that have a significant meaning on the effect of azelaic acid was CREB, ELK1 / SRF, STAT3, respectively cAMP / PKA, MAPK / ERK, IL-6 signaling pathways.

In addition, the gene expression of the inflammatory response mediators IL-6 and TNF-α in the hepatocyte hepa1c1c-7 of the mouse by the method of Example 7-4 and Example 7-5 by quantifying the qPCR Figure 7b Shown graphically. As shown in Figure 7b, it was confirmed that the IL-6 and TNF-α gene expression increased when the LPS treatment in the control group. In the azelaic acid treated group, it was confirmed that IL-6 and TNF-α gene expression was significantly reduced compared to the control group.

In addition, the method of Example 7-5 analyzed IL-6, TNFα, IL-1β inflammatory mediators, that is, inflammatory cytokines in Hepa1c1c-7 hepatocytes, and the results are shown in FIG. 7C. As can be seen in Figure 7c, the protein expression of IL-6 showed a tendency to decrease by azelaic acid.

The results indicate that Olfr544 activity promotes the activity of ELK1 / SRF, STAT3 and CREB in LPS-induced Hepa1c1c-7 cells, and azelaic acid reduces the gene expression of Il6 and Tnf in Hepa1c1c -7 cells and also increases IL- 6 suggests reducing the concentration of cytokines. This confirmed the potential anti-inflammatory function of azelaic acid through the activity of Olfr544 in cultured hepatocytes.

Example 8. Inflammation-Reducing Effect of Azelaic Acid in 3T3-L1 Cells

8-1) Cell Culture

Mouse adipocyte progenitors 3T3-L1 were purchased from Korea Cell Line Bank (Seoul, Korea). 3T3-L1 cells, 10% of heat-inactivated bovine calf selum using (Gibco, Grand island, New York, USA) and 1% PEST is (Dulbecco's modified Eagle's medium) was added DMEM of medium 5% CO _2, 37 ℃ Incubated under conditions. In order to differentiate 3T3-L1 adipocytes into adipocytes, 1 x 10 ⁶ cells per well were dispensed into ⁶ -well plates and cultured so that the cells were 100% dense. After 2 days, the cultured 3T3-L1 progenitor cells were treated with DMEM containing 10% FBS (fetal bovine serum) and MDI solution (0.5 mM IBMX, 0.5 μM dexamethasone, 10 μg / mL insulin). The medium was treated for 3 days. Subsequently, the treated cells were cultured in DMEM containing 10% FBS and 10 μg / mL insulin to confirm whether and how much lipid droplets were formed in the cells, thereby confirming the differentiation of the fat cells. .

8-2) Induction of inflammation and treatment of azelaic acid by LPS

Adipocytes 3T3-L1 cells were inoculated at a concentration of 1 × 10 ⁴ in 96 well-plates and cultured for 24 hours, followed by transfection of the construct containing the transcription factor promoter and luciferase for 8 hours. Experiments were divided into two groups: LPS-induced hepatocellular inflammation and no LPS treatment. The untreated control group treated with 50 μM of azelaic acid for 6 hours, and the experimental group induced inflammation with 1 hr of LPS 100 ng / mL, and then treated with 50 μM of azelaic acid for 6 hours. The sub-paths activated by are identified.

8-3) Ten Signal Path Experiments of GPCR

In order to confirm the mechanism of anti-inflammatory efficacy of azelaic acid, 10 signal transduction transcription factor analysis of GPCR was performed in the same manner as in Example 7-3.

8-4) Confirmation of Gene Expression of IL-6 and TNF-α

To confirm the gene expression of IL-6 and TNF-α, qRT-PCR was performed in the same manner as in Examples 7-4 and 7-5.

8-5) Confirmation of Inflammation-Reducing Effect of Azelaic Acid in 3T3-L1 Cells

The results of analyzing the transcription factor of the inflammation-related GPCR-dependent signaling pathway in the mouse adipocytes 3T3-L1 cells by the method of Example 8-3 are shown in Figure 8a. As shown in FIG. 8A, after inducing inflammation by treating LPS to 3T3-L1 cells, the transcription factor activity, which has a significant meaning on the effect of azelaic acid, was FOXO, which was involved in the PI-3 Kinase / AKT pathway signaling pathway. It is related.

In addition, the gene expression of inflammatory response mediators IL-6 and TNF-α in 3T3-L1 cells differentiated into adipocytes by the method of Example 8-4 was quantified, and is shown in FIG. 8B. . As shown in Figure 8b, it was confirmed that the expression of IL-6 gene significantly decreased in the azelaic acid treatment group. The results suggest that azelaic acid exhibits anti-inflammatory efficacy through reducing LPS-induced IL-6 inflammatory factor.

Example 9 Confirmation of Inflammation Inhibition Induced by LPS of Azelaic Acid in Mice

9-1) Experimental Animal Model

Male rats at week 8 of C57BL / 6J were purchased from Samtaco (Gyeonggi-do, Korea). All experiments were conducted according to the experimental method (Protocol No. KUIACUC-20090420-4) approved by Korea University Animal Experiment Committee. Mice were placed in photoperiod for 12 hours, at 21-25 ° C, and 50% -60% relative humidity. Normal and LPS-induced groups received 100 μL of tertiary distilled water per horse for 4 days using an oral zone and intraperitoneally injected 100 mg of LPS at a dose of 10 mg / kg 1 hour after tertiary distilled water on the last 4 days. It was. The mice treated with LPS after the administration of azelaic acid were also orally administered 100 mg / kg of azelaic acid per pig for 4 days, and 100 mg / kg LPS of 10 mg / kg LPS after 1 hour of azelaic acid in the last 4 days. Intraperitoneal injection. 16 hours after LPS intraperitoneal injection, anesthetized with avertin and mice were dissected to extract liver tissue. Liver tissue was collected and stored immediately in liquid nitrogen, and then stored at -80 ° C.

9-2) cDNA Synthesis

1 μg of RNA extracted from stored liver tissue was quantified and cDNA synthesized using ReverTra Ace® qPCR RT kit (TOYOBO, Osaka, Japan). More specifically, in order to increase the efficiency of the RT-PCR reaction, the extracted RNA was treated at 65 ° C. for 5 minutes and immediately stored on ice. Thereafter, a total of 8 μL of a reactant prepared with 2 μL of 4x DNA Master Mix, 0.5 μg RNA, and distilled water (nuclease-free water) containing a gDNA remover was reacted at 37 ° C. for 5 minutes. Then, 5x RT Master mix was added to the reaction product, and cDNA was synthesized by reacting for 37 ° C.-5 min, 50 ° C.-5 min, and 98 ° C.-5 min.

9-3) Quantitative real-time PCR (qPCR)

The cDNA synthesized by the above method was performed using a THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka, Japan) and an iQ5 iCycler system (Bio-rad, California, USA). More specifically, after denaturation at 95 ℃ for 4 minutes and 30 seconds, after further modification over 40 cycles (cycle) for 10 seconds at the same temperature, and then heated and cooled for 30 seconds at 55 ℃ ~ 60 ℃ Elongation was performed at 68 ° C. for 20 seconds. Each gene expression level was normalized using GAPDH expression. Primer designs were designed using nucleotideblast software from the National Center for Biotechnology Information (NCBI) and were purchased from Bionics (Seoul, Korea).

9-4) Statistical Processing

Statistical analysis was performed using the Student's t-test method, and the significance of P <0.05 was significant compared to the control group (*: P <0.05, **: P <0.01, ***: P < Means 0.001). One-way ANOVA was used for more than two groups. The error bars of each graph were represented by the mean ± SEM.

9-5) Confirmation of Inhibition of Inflammation Induced by LPS of Azelaic Acid in Mice

In order to investigate the effect of AzA on inflammation in mouse in vivo in Example 9-1, C57BL / 6J male mice were intraperitoneally infused with AzA for 3 days and then injected with LPS. And the expression levels of inflammatory cytokines, hepatokines, growth factors, and inflammatory marker genes were analyzed by the method of 9-3. As a result, as shown in Figure 9, the gene expression level of the inflammatory cytokines IL-6 and TNF-α in A shows a tendency to reduce the inflammatory response by azelaic acid after inflammation is induced by LPS. A is the level of gene expression of inflammatory markers, B is the level of gene expression of growth factors, and C and D is the level of gene expression of hepatokines.

The above description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

<110> Korea University Industry and Academy Cooperation Foundation <120> Composition for preventing, treating, or improving fatty liver          compring of azelaic acid as an active ingredient <130> MP19-038 <150> KR 10-2018-0020080 <151> 2018-02-20 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide encoding shRNA hairpin sequences targeting          Olfr544 <400> 1 caccgctcac tgttcgcatc ttcattcgaa aatgaagatg cgaacagtga g 51 <210> 2 <211> 47 <212> DNA <213> Artificial Sequence <220> Oligonucleotide encoding non-targeting scrambled shRNA hairpins <400> 2 caccgtaagg ctatgaagag ataccgaagt atctcttcat agcctta 47

Claims (10)

Azelaic acid as an active ingredient, fatty liver prevention or treatment pharmaceutical composition. The method of claim 1,
The composition is characterized in that to activate Olfr544 (Olfactory receptor 544) in liver tissue, fatty liver prevention or treatment pharmaceutical composition. The method of claim 1,
The composition is characterized in that to activate the peroxisome proliferator-activated receptor α (PPARα) in liver tissue, fatty liver prevention or treatment pharmaceutical composition. The method of claim 1,
The composition is characterized in that to inhibit the accumulation of triglycerides in liver tissue, fatty liver prevention or treatment pharmaceutical composition. The method of claim 1,
The fatty liver is characterized in that non-alcoholic fatty liver, fatty liver prevention or treatment pharmaceutical composition. Food composition for preventing or improving fatty liver, comprising azelaic acid as an active ingredient. The method of claim 6,
The composition is characterized in that to activate Olfr544 (Olfactory receptor 544) in liver tissue, fatty liver prevention or improvement of food composition. The method of claim 6,
The composition is characterized in that to activate the peroxisome proliferator-activated receptor α (PPARα) in liver tissue, fatty liver prevention or improvement for food composition. The method of claim 6,
The composition is characterized in that to inhibit the accumulation of triglycerides in liver tissue, fatty liver prevention or improvement for food composition. The method of claim 6,
The fatty liver is characterized in that non-alcoholic fatty liver, fatty liver prevention or improvement food composition.

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