WO2024128869A1 - Pharmaceutical composition for ameliorating, preventing, or treating fatty liver disease, comprising methyl linoleic acid - Google Patents

Abstract

The present invention relates to a pharmaceutical composition that can effectively inhibit or prevent fat accumulation in hepatocytes even at a low concentration thereof that does not cause cytotoxicity, and thereby has excellent effects in preventing, ameliorating, or treating various fatty liver diseases.

Description

Pharmaceutical composition for improving, preventing or treating fatty liver disease containing methyl linoleic acid

The present invention relates to a pharmaceutical composition for improving, preventing or treating fatty liver disease containing methyl linoleic acid or a pharmaceutically acceptable salt thereof.

Fatty liver disease is a disease caused by excessive accumulation of fat in the liver. It refers to cases where more than 5% of the liver weight is fat, especially neutral fat. It is known to be caused by excessive alcohol consumption or drug use, hepatitis virus, some genetic diseases, and metabolic diseases such as obesity or type 2 diabetes.

Among these, those caused by alcohol consumption are classified as alcoholic fatty liver, and those caused by alcohol consumption are classified as non-alcoholic fatty liver. It is a disease that starts from simple steatosis and progresses to steatohepatitis, liver cirrhosis, etc.

There is no clear treatment for fatty liver other than improving lifestyle habits such as diet and exercise. Diabetes drugs (thiazolidinediones, metformin, GLP-1 agonist, DPP4 inhibitor), antioxidants and hepatocyte protectors (vitamin E, ursodeoxycholic acid), and non-steroidal anti-inflammatory drugs. (NSAID) etc. are used in a limited manner.

Currently, the development of treatments for fatty liver/steatohepatitis using various new molecular targets is active, with Phase 3 clinical trials underway for Elafibranor, a PPARα/δ ligand, Cenicriviroc, a CCR2/5 inhibitor, Obeticholic acid, an FXR agonist, and Selonsertib, an ASK-1 inhibitor. It is progressing (Nature Medicine, 2018, 24(7):908-922).

Meanwhile, methyl linoleate is a methyl ester derivative of linoleic acid and is known to be found in many types of animal fats, soybean oil, babassu oil, flaxseed oil, corn oil, and poppy seed oil.

Recently, the antioxidant and anticancer effects of methyl linoleic acid were reported (An Acad Bras Cienc, 2017 Jul-Sep;89(3):1671-1681. doi: 10.1590/0001-3765201720160908. Epub 2017 Aug 31).

However, to date, not much research has been conducted on the physiological activity of methyl linoleic acid, and in particular, no research has been conducted on its effect in suppressing fat accumulation in hepatocytes.

The present invention is intended to solve the above problems, and its purpose is to provide a pharmaceutical composition for preventing, improving, or treating fatty liver disease, which contains methyl linoleate as an active ingredient.

Additionally, the purpose of the present invention is to provide a health functional food composition for preventing or improving fatty liver disease, comprising methyl linoleic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

Additionally, the purpose of the present invention is to provide a feed composition for preventing or improving fatty liver disease, comprising methyl linoleic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

Additionally, the purpose of the present invention is to provide a cosmetic composition for preventing, improving, or treating fatty liver disease, comprising methyl linoleic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the purpose of the present invention is to provide a method for preventing, improving or treating fatty liver disease, comprising administering to an individual a pharmaceutical composition containing methyl linoleic acid or a pharmaceutically acceptable salt thereof as an active ingredient. Do it as

The pharmaceutical composition for preventing, improving or treating fatty liver disease of the present invention for achieving the above object is characterized by containing methyl linoleate or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the health functional food composition for preventing or improving fatty liver disease of the present invention is characterized by containing methyl linoleic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the feed composition for preventing or improving fatty liver disease of the present invention is characterized by containing methyl linoleic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the cosmetic composition for preventing, improving or treating fatty liver disease of the present invention is characterized by containing methyl linoleic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the method for preventing, improving or treating fatty liver disease of the present invention is characterized by comprising administering to a subject a pharmaceutical composition containing methyl linoleic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

The pharmaceutical composition of the present invention contains methyl linoleate or a pharmaceutically acceptable salt thereof as an active ingredient, and thus can effectively inhibit or prevent fat accumulation in hepatocytes even at low concentrations that do not cause cytotoxicity, thereby It has the advantage of being effective in preventing, improving, or treating various fatty liver diseases.

The health functional food composition of the present invention has the same advantages as described above by containing methyl linoleic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

The feed composition of the present invention has the same advantages as described above by containing methyl linoleic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

The cosmetic composition of the present invention has the same advantages as described above by containing methyl linoleic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

Figure 1 is a diagram showing the experimental results of Experimental Example 1 of the present invention. Figure 1 (A) is the result of observing the cell survival rate according to treatment with palmitic acid (PA) at each concentration through a phase contrast microscope, and Figure 1 (B) is the result of observing the cell survival rate through MTT assay. am.

Figure 2 is a diagram showing the results of experiment 2 of the present invention. Figure 2 (A) shows the results of Oil Red O staining of liver cells according to treatment with palmitic acid (PA) at each concentration, and Figure 2 (B) is a schematic quantification of the Oil Red O staining results. , Figure 2 (C) shows the staining results of BODIPY 493/503, Hoechst 33342, and Merge (merging the results of BODIPY 493/503 and Hoechst 33342) of hepatocytes according to treatment with palmitic acid (PA) at each concentration. 2 (D) is a diagram quantifying the results of the BODIPY 493/503 staining.

Figure 3 is a diagram showing the experimental results of Experimental Example 3 of the present invention. Figure 3 (A) shows the results of Oil Red O staining of liver cells according to treatment with methyl linoleic acid at each concentration (where Control is the control group not treated with either palmitic acid (PA) or methyl linoleic acid). Figure 3 (B) is a schematic quantification of the Oil Red O staining results, Figure 3 (C) is the results of the liver cell toxicity test according to methyl linoleic acid treatment at each concentration, and Figure 3 (D) is the results of each concentration. This is the result of BODIPY 493/503 staining of hepatocytes following treatment with methyl linoleic acid, and Figure 3 (E) is a diagram quantifying the results of the BODIPY 493/503 staining.

Figure 4 is a diagram showing the experimental results of Experimental Example 4 of the present invention. Figure 4 (A) shows the BODIPY 493/503 staining results of hepatocytes following treatment with conjugated linoleic acid at each concentration (where Control is the control group not treated with either palmitic acid (PA) or conjugated linoleic acid). Figure 4 (B) is a diagram quantifying the results of the BODIPY 493/503 staining, and Figure 4 (C) is the results of a hepatocellular toxicity test according to conjugated linoleic acid treatment at each concentration.

Figure 5 is a diagram showing the experimental results of Experimental Example 5 of the present invention. Figure 5 (A) shows the BODIPY 493/503 staining results of hepatocytes according to methyl linolenic acid treatment at each concentration (where Control is the control group not treated with either palmitic acid (PA) or methyl linolenic acid). Figure 5 (B) is a diagram quantifying the results of the BODIPY 493/503 staining, and Figure 5 (C) is the results of a hepatocellular toxicity test according to methyl linolenic acid treatment at each concentration.

In the present invention, when a part "includes" a certain component, this means that it may further include other components rather than excluding other components, unless specifically stated to the contrary.

In the present invention, “pharmaceutically acceptable salt” refers to a concentration of methyl linoleic acid that is relatively non-toxic and harmless to the individual receiving it and has an effective effect, and the side effects caused by this salt do not reduce the beneficial effects of methyl linoleic acid. means any and all organic or inorganic addition salts.

In the present invention, “foodologically acceptable salt” can be defined in the same way as the pharmaceutically acceptable salt.

In the present invention, “feedologically acceptable salt” may be defined in the same way as the pharmaceutically acceptable salt.

In the present invention, “feed” means any natural or artificial diet, meal, etc. that is suitable for or for animals to eat, ingest, and digest.

In the present invention, “prevention” refers to any action that inhibits fat accumulation in liver cells or delays the progression of fatty liver disease by using the composition of the present invention.

*32 In the present invention, “improvement” or “treatment” means suppressing fat accumulation in hepatocytes using the composition of the present invention, or any action that improves or beneficially changes fatty liver disease as a result, and is clinically effective. It refers to an attempt to obtain a useful result or desirable result, including results. A useful or desirable clinical outcome may be alleviation or improvement of one or more symptoms or conditions, reduction of the extent of the disease, stabilization of the disease state, inhibition of the development of the disease, inhibition of the spread of the disease, or delay or slowing of the progression of the disease, whether detectable or not. , may include, but is not necessarily limited to, delaying or delaying the onset of a disease, improving or alleviating a disease state, and reducing (partial or total). Additionally, “improvement” or “treatment” can mean prolonging the patient's survival beyond what would be expected in the absence of treatment. Additionally, “improvement” or “treatment” may refer to inhibiting the progression of a disease, temporarily slowing the progression of the disease, or more preferably permanently halting the progression of the disease. In the present invention, it may mean improving the survival of patients by improving the treatment of fatty liver disease, especially non-alcoholic fatty liver disease.

In the present invention, “individual” refers to all animals, including humans, that have already developed or may develop fatty liver disease. For example, the individual may be a dog, cow, horse, rabbit, mouse, rat, chicken, or It may refer to all mammals, including humans, but is not limited thereto, and the entity may be an entity other than humans.

Hereinafter, the present invention will be described in more detail.

The pharmaceutical composition according to one aspect of the present invention contains methyl linoleate or a pharmaceutically acceptable salt thereof as an active ingredient, so that it can effectively inhibit or prevent fat accumulation in hepatocytes even at low concentrations that do not cause cytotoxicity. This has the advantage of being effective in preventing, improving, or treating various fatty liver diseases.

The methyl linoleate may be a compound represented by the following formula (1).

[Formula 1]

The methyl linoleic acid is a methyl ester derivative and is a substance found in various types of animal fat, soybean oil, babassu oil, linseed oil, corn oil, poppy seed oil, etc. The methyl linoleic acid can be used as a substrate to measure the antioxidant effect of a specific substance against lipid peroxidation, and has the advantage of having an excellent therapeutic effect for essential fatty acid deficiency syndrome. In addition, the methyl linoleic acid has the advantage of being excellent in suppressing or preventing fat accumulation in liver cells.

The pharmaceutical composition according to one embodiment of the present invention may inhibit fat accumulation in hepatocytes.

According to one embodiment of the present invention, the fat accumulation may be induced by free fatty acid. The free fatty acids are also called non-ester fatty acids, and their main components include palmitic acid, stearic acid, and oleic acid, and can bind to albumin in the blood and be transported interstitially. When adipocyte dysfunction occurs due to causes such as unbalanced diet and obesity, free fatty acids may abnormally accumulate in liver tissue due to lipolysis, which may increase the incidence and risk of fatty liver disease. there is. Preferably, the fat accumulation may be induced by palmitic acid, but is not limited thereto.

According to one embodiment of the present invention, the fatty liver disease may include alcoholic fatty liver disease or non-alcoholic fatty liver disease.

The fatty liver disease refers to a disease induced by fatty liver, and fatty liver refers to a phenomenon in which neutral fat appears abnormally deposited within liver cells, unlike normal cases. In the case of a normal liver, approximately 5% is composed of adipose tissue, and neutral fat, fatty acids, phospholipids, cholesterol, and cholesterol ester are the main components of fat, but once fatty liver occurs, most of the components are replaced by neutral fat, and the amount of neutral fat decreases. If it is more than 5% of the liver weight, fatty liver is diagnosed. The fatty liver disease is caused by disorders in fat metabolism within hepatocytes or defects in the process of transporting excess fat.

The alcoholic fatty liver refers to a case where fat synthesis is promoted in the liver due to excessive consumption of alcohol and normal energy metabolism does not occur. Alcohol cannot be stored in the body and is completely eliminated through oxidation in the liver. Specifically, in the liver, alcohol is largely divided into the alcohol dehydrogenase (ADH) pathway and the microsomal alcohol oxidizing system (MEOS) pathway. It is metabolized by three pathways including the catalase pathway and converted into acetaldehyde, which is then metabolized into acetate salt by aldehyde dehydrogenase (ALDH). At this time, the acetaldehyde is toxic and can damage liver cells, and in the microsomal alcohol oxidation system pathway, superoxide (O ₂ ), it is known that reactive oxygen species (ROS), such as hydrogen peroxide (H ₂ O ₂ ) and peroxynitrite, are generated and cause oxidative stress, causing liver damage. In addition, as a result of the metabolism of alcohol, a large amount of fatty acids are produced, causing fat to accumulate in the liver, and fatty liver accumulated in the liver for this reason is called alcoholic fatty liver.

The non-alcoholic fatty liver disease refers to a case of fatty liver disease without a history of alcohol consumption, and is known to be related to metabolic diseases such as obesity, diabetes, and hyperlipidemia. According to one embodiment of the present invention, the non-alcoholic fatty liver disease may include one or more selected from the group consisting of non-alcoholic simple fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic cirrhosis, and end-stage fibrotic liver disease.

The non-alcoholic steatohepatitis refers to a case where inflammation of a more severe degree occurs among patients with non-alcoholic fatty liver disease. If treatment is delayed, it may progress to cirrhosis and hepatocellular carcinoma.

The non-alcoholic cirrhosis is also called liver cirrhosis and refers to all forms of liver disease characterized by loss of a significant amount of liver cells, proliferation of fibrous tissue, and regenerative nodules. The cirrhosis can also be induced by insulin resistance, and can cause complications such as ascites (a symptom of fluid in the stomach), varicose bleeding, and hepatic coma.

According to one embodiment of the present invention, the concentration of the methyl linoleic acid or a pharmaceutically acceptable salt thereof may be 0.1 to 100 μM, preferably 10 to 50 μM, and more preferably 10 to 20 μM. You can. When the concentration of methyl linoleic acid or its pharmaceutically acceptable salt contained in the pharmaceutical composition satisfies the above-mentioned range, not only is the effect of inhibiting fat accumulation in hepatocytes improved, but also cytotoxicity to hepatocytes is not observed or is very low. It has the advantage of excellent stability. If the concentration of methyl linoleic acid or a pharmaceutically acceptable salt thereof is less than the above range, the effect of inhibiting fat accumulation in hepatocytes may be reduced, and if it exceeds the above range, cytotoxicity may be enhanced.

The pharmaceutical composition 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, and sterile injection solutions according to conventional methods. Carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, and cellulose. , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is 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. These solid preparations include the compound of the present invention with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, It is prepared by mixing gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. . Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used.

In addition to the above-mentioned active ingredients, the pharmaceutical composition may further include any compounds or natural extracts known to have activities whose safety has already been verified in order to increase and reinforce the effect of inhibiting fat accumulation in hepatocytes.

The present invention provides a pharmaceutical drug containing hepatocytes induced by fatty liver or hepatocytes induced by fat accumulation, and the methyl linoleic acid or a pharmaceutically acceptable salt thereof as active ingredients in vitro or ex vivo. A method for inhibiting fat accumulation in hepatocytes is provided, comprising the step of contacting the composition.

The present invention provides a method for preventing, improving, or treating fatty liver disease, comprising administering to an individual a pharmaceutical composition containing methyl linoleic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention provides a method for inhibiting fat accumulation in hepatocytes, comprising administering to a subject a pharmaceutical composition containing methyl linoleic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

The method of administering the pharmaceutical composition to an individual is not particularly limited in the present invention, but may be administered, for example, orally, rectally, or by intravenous, intramuscular, subcutaneous, intrauterine dura, or intracerebrovascular injection. there is.

The dosage of the pharmaceutical composition may vary depending on the age, gender, and weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological state, the route of administration, and the judgment of the prescriber. No restrictions. Dosage determination based on these factors is within the level of one skilled in the art and may range, for example, from 0.01 mg/kg/day to approximately 2000 mg/kg/day, preferably from 0.1 mg/kg/day to 1000 mg/kg/day. It may be mg/kg/. The above administration may be administered once a day or may be administered several times, but this is also not particularly limited in the present invention.

Another aspect of the present invention includes the above-described methyl linoleic acid or a food-acceptable salt thereof as an active ingredient, thereby effectively inhibiting or preventing fat accumulation in hepatocytes even at low concentrations that do not cause cytotoxicity, thereby preventing various fatty liver diseases. It relates to a food composition for preventing or improving fatty liver disease, which has excellent prevention or improvement effects.

The food composition can be manufactured in all food forms, such as functional foods, nutritional supplements, health foods, and food additives, for example, and can be manufactured in the various forms. Foods can be manufactured in a variety of ways according to common methods known in the art, and the present invention does not specifically limit this.

For example, as the health food, the food composition itself can be prepared and consumed in the form of tea, juice, or drink, or can be granulated, encapsulated, or powdered for consumption. In addition, the functional foods include beverages (including alcoholic beverages), fruits or processed foods thereof (e.g. canned fruit, bottled foods, jam, marmalade, etc.), fish, meat or processed foods thereof (e.g. ham, sausage, corned beef, etc.), Bread or noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, retort food, It can be manufactured by adding separate extracts to frozen foods and various seasonings (e.g. soybean paste, soy sauce, sauce, etc.). Additionally, in order to use the food composition of the present invention in the form of a food additive, it can be prepared and used in the form of a powder or concentrate.

In order to be manufactured into various types of food as described above, the food composition may further include foodologically acceptable carriers or additives in addition to the above-mentioned active ingredients, and in the manufacture of the dosage form or form to be manufactured, in the art Any carriers or additives known to be usable may be further included without particular limitation.

Another aspect of the present invention includes the methyl linoleic acid or a feed-acceptable salt thereof as an active ingredient, thereby effectively inhibiting or preventing fat accumulation in hepatocytes even at low concentrations that do not cause cytotoxicity, thereby preventing various fatty liver diseases. It relates to a feed composition for preventing or improving fatty liver disease, which has excellent prevention or improvement effects.

The feed composition includes, for example, plant feed such as grains, roots and fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, cucurbits or grain by-products; Examples include, but are not limited to, animal feeds such as proteins, minerals, fats and oils, minerals, single-cell proteins, zooplanktons, or foods, and may refer to feeds commonly used in the industry without particular limitation. You can.

The form of the feed composition may include powder feed, solid feed, moist pellet feed, dry pellet feed, EP (extruder pellet) feed, raw feed, etc., but is not limited thereto.

The feed composition may further contain binders, emulsifiers, preservatives, etc. in addition to the above-mentioned active ingredients for the purpose of preventing quality degradation, etc., and to increase effectiveness, amino acids, vitamins, enzymes, flavoring agents, non-protein nitrogen compounds, It may further include additives such as silicate agents, buffering agents, extractants, oligosaccharides, and feed mixtures, but is not limited thereto.

Another aspect of the present invention includes the methyl linoleic acid or a pharmaceutically acceptable salt thereof as an active ingredient, thereby effectively inhibiting or preventing fat accumulation in hepatocytes even at low concentrations that do not cause cytotoxicity, thereby preventing various fatty liver diseases. It relates to a cosmetic composition for preventing or improving fatty liver disease, which has excellent prevention, improvement or treatment effects.

The formulation of the cosmetic composition is not particularly limited in the present invention, but includes, for example, softening lotion, astringent lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing cream. It can be formulated into cosmetics such as water, pack, powder, body lotion, body cream, body oil, or body essence, and can also be applied in a form that is applied to the skin or absorbed into the skin using a microneedle, etc.

The cosmetic composition includes, as carriers permitted in the cosmetics process, hydrocarbons such as petrolatum, liquid paraffin, and gelled hydrocarbons (aka: plastibase); Animal and vegetable oils such as medium-chain fatty acid triglycerides, pork fat, hard fat, and cocoa fat; higher fatty alcohols and fatty acids and their esters such as cetanol, stearyl alcohol, stearic acid, and isopropyl palmitate; Water-soluble bases such as macrogol (polyethylene glycol), 1,3-butylene glycol, glycerol, gelatin, white sugar, and sugar alcohol; Emulsifiers such as glycerin fatty acid ester, polyoxyl stearate, and polyoxyethylene hydrogenated castor oil; Adhesives such as acrylic acid ester and sodium alginate; Propellants such as liquefied petroleum gas and carbon dioxide; It may further contain preservatives such as paraoxybenzoic acid esters, and in addition to these, stabilizers, flavoring agents, colorants, pH adjusters, diluents, surfactants, preservatives, antioxidants, etc. may be further included as needed.

The cosmetic composition may be prepared in the form of an external preparation according to a conventional method including the above-described components. The use of the external agent may be applied to a local wound by a conventional method.

Hereinafter, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are merely illustrative of the present invention, and it is obvious to those skilled in the art that various changes and modifications are possible within the scope and technical spirit of the present invention. , it is natural that such variations and modifications fall within the scope of the attached patent claims. In the following examples and comparative examples, “%” and “part” indicating content are based on weight, unless otherwise specified.

Examples and Comparative Examples

Example

As an example, methyl linoleate (Tokyo Chemical Co., cat#: s0325) was prepared.

Comparative Example 1

As Comparative Example 1, conjugated linoleic acid (CLA, manufactured by Sigma Aldrich, cat#: O-5507) was prepared.

Comparative Example 2

*90 As Comparative Example 2, methyl linolenate (methyl linolenate, manufactured by Sigma Aldrich, cat#: L-2626) of the following formula (2) was prepared.

[Formula 2]

Preparation example: Preparation of palmitic acid

A NaCl solution of pH 7.4 was prepared using 150mM NaCl. 0.68mM of BSA (Bovine serum albumin, manufactured by Sigma Aldrich, cat#: A7030) was dissolved in the NaCl solution, and a final 0.34mM BSA solution was prepared and used as a control.

0.68mM BSA (manufactured by Sigma Aldrich, cat#: A7030) and 5mM sodium palmitate (manufactured by Sigma Aldrich, cat#: P-9767) were dissolved in 150mM NaCl and incubated at 37°C (±3). ℃) for 1 hour, and finally 2mM palmitic acid (PA) was synthesized.

The BSA solution and palmitic acid (PA) solution of the control group were respectively filtered and stored in a freezer at -20°C before use.

Experimental Example 1: Confirmation of hepatocellular toxicity by palmitic acid

Palmitic acid (PA) bound to BSA (bovine serum albumin) prepared in the above production example was treated with different concentrations (10 μM, 20 μM, 50 μM, 100 μM, 200 μM, 500 μM) to AML12 mouse liver cells. , After culturing for 24 hours, the survival rate of cells was observed through phase contrast microscopy (see (A) in Figure 1) and MTT assay (see (B) in Figure 1).

Referring to Figure 1 of the present invention, palmitic acid did not show any significant cytotoxicity to hepatocytes up to a concentration of 100 μM, but it was confirmed that it showed significant cytotoxicity at concentrations higher than that. ***p < 0.001 (vs control, palmitic acid concentration 0μM).

Experimental Example 2: Confirmation of fat accumulation in hepatocytes by palmitic acid

Palmitic acid (PA) bound to BSA (bovine serum albumin) prepared in the above preparation example was treated at various concentrations (20 μM, 50 μM, 100 μM) to AML12 mouse liver cells, and cultured for 24 hours. The degree of fat accumulation in the hepatocytes was confirmed using Oil Red O staining (see (A) and (B) of Figure 2) and BODIPY 493/503 staining (see (C) and (D) of Figure 2). Additionally, the number of living cells was confirmed by staining the cell nuclei using Hoechst 33342 (see (C) in Figure 2). ***p < 0.001 (vs control, palmitic acid concentration 0μM).

Referring to Figure 2 of the present invention, it was confirmed that fat accumulation was possible in a concentration-dependent manner by treatment with palmitic acid, and through this, in vitro experimental conditions for fat accumulation in hepatocytes using palmitic acid were established. It was built.

Experimental Example 3: Confirmation of hepatocellular toxicity and fat accumulation inhibition effect by methyl linoleic acid

As confirmed in Experimental Example 2, under the PA 50μM condition, which is one of the experimental conditions in which fat accumulates in mouse hepatocyte AML12, methyl linoleic acid of the above example was treated at different concentrations (10μM, 20μM), and then 24 hours later, fat The degree of accumulation was stained with Oil Red O (see (A) in Figure 3), and the results were quantified (see (B) in Figure 3). ** p < 0.01, *** p < 0.001. Under the same conditions (PA 50 μM), the methyl linoleic acid of the above example was treated at different concentrations (20 μM, 50 μM), and then the degree of fat accumulation was measured 24 hours later using BODIPY 493/503 staining (see (D) in Figure 3). This was confirmed through, and the results were quantified (see (E) in Figure 3). ***p < 0.001.

In addition, in order to confirm the cytotoxicity of methyl linoleic acid in the above example, untreated mouse hepatocytes AML12 were treated with methyl linoleic acid in each concentration (1 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM) for 24 hours. Cytotoxicity after culturing for a period of time was confirmed by MTT assay (see (C) in Figure 3).

Referring to Figure 3, it was confirmed that when treated with methyl linoleic acid of the present invention, fat accumulation in hepatocytes was significantly reduced (see Figures 3 (A), (B), (D), and (E). Referring to Figure 3 (C), it was confirmed that methyl linoleic acid of the present invention does not exhibit toxicity to hepatocytes.

Experimental Example 4: Conjugated linoleic acid Confirmed effect of inhibiting hepatocellular toxicity and fat accumulation

As confirmed in Experimental Example 2, under the PA 50μM condition, which is one of the experimental conditions for fat accumulation in mouse hepatocyte AML12, conjugated linoleic acid (CLA) of Comparative Example 1 was administered at each concentration (10μM, 50μM). ), and 24 hours later, fat accumulation was stained with BODIPY 493/503 (see (A) in Figure 4), and the results were quantified (see (B) in Figure 4). ** p < 0.01, *** p < 0.001.

In addition, in order to confirm the cytotoxicity of the conjugated linoleic acid of Comparative Example 1, untreated mouse hepatocytes AML12 were treated with the conjugated linoleic acid of Comparative Example 1 at different concentrations (1 μM, 5 μM, 10 μM, 50 μM, 100 μM) Cell toxicity after culturing for 24 hours was confirmed by MTT assay (see (C) in Figure 4).

Referring to Figure 4, it was confirmed that even though conjugated linoleic acid, which has a similar parent structure to the methyl linoleic acid of the present invention, does not have an effect of suppressing fat accumulation when not treated or when treated with fat conjugated linoleic acid, unlike methyl linoleic acid. Rather, it was confirmed that as the concentration of conjugated linoleic acid increased, fat accumulation was not suppressed but increased (see Figure 4 (A) and (B)). In addition, it was confirmed that the conjugated linoleic acid of Comparative Example 1 exhibited cytotoxicity to hepatocytes, unlike the methyl linoleic acid of the present invention (see Figure 4 (C)).

Experimental Example 5: Confirmation of hepatocellular toxicity and fat accumulation inhibition effect by methyl linolenic acid

As confirmed in Experimental Example 2, methyl linolenate of Comparative Example 2 was treated at each concentration (10μM, 50μM) under the PA 50μM condition, which is one of the experimental conditions for fat accumulation in mouse hepatocytes AML12. Then, 24 hours later, fat accumulation was stained with BODIPY 493/503 (see (A) in Figure 5), and the results were quantified (see (B) in Figure 5). ***p < 0.001.

In addition, in order to confirm the cytotoxicity of methyl linolenic acid in Comparative Example 2, untreated mouse hepatocytes AML12 were treated with methyl linolenic acid in Comparative Example 2 at different concentrations (5 μM, 10 μM, 20 μM, 50 μM, 100 μM) Cell toxicity after culturing for 24 hours was confirmed by MTT assay (see (C) in Figure 5).

Referring to Figure 5, it was confirmed that even with methyl linolenic acid, which has a similar structure to the methyl linoleic acid of the present invention, unlike methyl linoleic acid, there was no effect of suppressing fat accumulation when not treated with methyl linolenic acid or when treated with methyl linolenic acid. Rather, the conjugated It was confirmed that as the concentration of linoleic acid increased, fat accumulation was not suppressed but increased (see Figures 5 (A) and (B)). Meanwhile, it was confirmed that the methyl linolenic acid did not exhibit toxicity to hepatocytes (see (C) in Figure 5).

Claims (10)

1. A pharmaceutical composition for preventing, improving, or treating fatty liver disease, comprising methyl linoleate or a pharmaceutically acceptable salt thereof as an active ingredient.
2. According to paragraph 1,

   A pharmaceutical composition, wherein the methyl linoleic acid inhibits fat accumulation in hepatocytes.
3. According to paragraph 2,

   A pharmaceutical composition, wherein the fat accumulation is induced by free fatty acids.
4. According to paragraph 1,

   A pharmaceutical composition, wherein the fatty liver disease includes alcoholic fatty liver disease or non-alcoholic fatty liver disease.
5. According to clause 4,

   A pharmaceutical composition, wherein the non-alcoholic fatty liver disease includes one or more selected from the group consisting of non-alcoholic simple fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic cirrhosis, and end-stage fibrotic liver disease.
6. According to paragraph 1,

   A pharmaceutical composition, characterized in that the concentration of the methyl linoleic acid or a pharmaceutically acceptable salt thereof is 0.1 to 100 μM.
7. A food composition for preventing or improving fatty liver disease, comprising methyl linoleic acid or a foodologically acceptable salt thereof as an active ingredient.
8. A feed composition for preventing or improving fatty liver disease, comprising methyl linoleic acid or a feed-acceptable salt thereof as an active ingredient.
9. A cosmetic composition for preventing, improving, or treating fatty liver disease, comprising methyl linoleic acid or a pharmaceutically acceptable salt thereof as an active ingredient.
10. A method for preventing, improving, or treating fatty liver disease, comprising: administering to an individual a pharmaceutical composition containing methyl linoleic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

Images