KR20230155763A - Composition for improving lipid metabolism comprising Patulin, Dianemycin or Arnicolide D - Google Patents

Abstract

The present invention relates to a composition for improving lipid metabolism or for preventing, improving or treating metabolic diseases related to lipid accumulation, containing patulin, dianemycin or arnicolide D as an active ingredient. will be.
Patulin, dianemycin or arnicolide D of the present invention improves blood lipid concentration by suppressing lipid accumulation and has the effect of preventing, improving or treating metabolic diseases related to lipid accumulation, and can be used as a material for medicines, foods, etc. It can be utilized.

Description

Composition for improving lipid metabolism comprising Patulin, Dianemycin or Arnicolide D}

The present invention relates to a composition for improving lipid metabolism containing patulin, dianemycin or arnicolide D.

Adipose tissue, or body fat, is loose connective tissue composed mostly of fat cells. Adipose tissue contains, in addition to adipocytes, a stromal vascular fraction (SVF) of cells such as preadipocytes, fibroblasts, vascular endothelial cells, and various immune cells (i.e., adipose tissue macrophages (ATMs)). Contains Adipose tissue is derived from preadipocytes. The main role of adipose tissue is to store energy in the form of lipids, while also protecting and insulating the body from shock. However, excessive fat accumulation in adipose tissue is undesirable, primarily causes aesthetic problems, and may ultimately cause pathological conditions such as obesity, lipid storage disease, and hyperlipidemia.

On the other hand, if cholesterol is properly regulated in the body, cholesterol accumulation does not occur in plasma and tissues, but if the level of cholesterol in the body increases, hypercholesterolemia occurs, and abnormalities in cholesterol metabolism regulation occur, leading to low-density lipoprotein-cholesterol. Lipoprotein components such as (LDL-cholesterol) and high-density lipoprotein-cholesterol (HDL-cholesterol) change quantitatively. In particular, low-density lipoprotein-cholesterol (LDL-cholesterol) among blood cholesterol is known to be a risk factor for cardiovascular disease, and an increase in blood triglyceride concentration lowers the concentration of high-density lipoprotein-cholesterol (HDL-cholesterol) and triglyceride It is known to be a major cause of coronary artery disease by increasing the content of chylomicron residues, which are lipoprotein particles mainly composed of triglyceride. In addition, blood lipids are a major cause of arteriosclerosis because they pass through vascular endothelial cells better than other lipids, and a rapid rise in triglycerides after a meal is known to be closely related to the occurrence of stroke. On the other hand, HDL-cholesterol has the effect of removing cholesterol from blood vessel walls and is known as a preventive factor for cardiovascular disease. Therefore, in order to prevent or treat various diseases caused by abnormalities in lipid metabolism, each blood lipid composed of triglyceride, cholesterol, phospholipids, and free fatty acid in vivo is analyzed. It can be said that it is important to maintain the ingredients at normal concentrations.

Currently, the treatment methods used to prevent and treat lipid metabolism diseases by appropriately controlling the concentration of lipid components in the blood mainly include dietary fiber, saponin, vitamin C, vitamin E, and essential oils. A diet that recommends the intake of essential amino acids, etc. is being used, but there is a problem with diet therapy that is difficult to maintain for a long period of time because it can be accompanied by mental illness due to dietary restrictions. In addition to the above-mentioned diet, drug therapy is used to treat lipid metabolism diseases. Such lipid-lowering drugs must be administered for life, and there is a risk of complications such as increased liver function and muscle disease, and in particular, liver function levels are lowered. There is a problem that drug treatment is difficult when the increase is more than 3 times, so there is a need for useful active ingredients among natural ingredients that can treat lipid-related cardiovascular disease and obesity.

In addition, currently used obesity treatments include appetite suppressants, heat metabolism promoters, digestion and absorption inhibitors, and hormone regulators that are effective by reducing food intake, controlling body metabolism, and increasing body heat response. Appetite suppressants include adrenergic drugs, serotonergic drugs, and adrenergic-serotonin drugs, but among these, serotonergic drugs are banned due to side effects of heart valve disease. Heat metabolism accelerators include ephedrine-caffeine complexes and adipocyte 3 receptor agonists, and digestion and absorption inhibitors include lipase inhibitors such as Xenical and a low-fat food additive called Olestra. Many drugs used in drug treatment to date have been reported to have side effects such as yo-yo phenomenon, anorexia, headache, increased blood pressure, steatorrhea, diarrhea, abdominal distension, and abdominal pain. It is interpreted as a result of unverified products. Therefore, the development of new natural materials using herbal medicine resources, which have been proven to be safe with improved obesity treatment effectiveness and fewer side effects, is a task that is continuously required in today's era.

Under this background, the present inventors conducted research using patulin, dianemycin, and arnicolide D derived from natural products and confirmed that they are effective in suppressing lipid accumulation, improving blood lipid concentration, and preventing or treating lipid-related diseases. and completed the present invention.

European Patent No. 2201957 B1 European Patent No. 0117107 B1

European Journal of Pharmacology, Volume 795, 15 January 2017, Pages 169-178

The object of the present invention is to provide a composition for improving lipid metabolism or for preventing, improving or treating lipid-related metabolic diseases containing patulin, dianemycin or arnicolide D as an active ingredient. It is provided.

Another object of the present invention is to provide a food composition for improving lipid metabolism or preventing lipid-related metabolic diseases containing patulin, dianemycin or arnicolide D as an active ingredient.

Another object of the present invention is to provide a health functional food for improving lipid metabolism or preventing lipid-related metabolic diseases containing patulin, dianemycin or arnicolide D as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing, improving or treating lipid-related metabolic diseases containing patulin, dianemycin or arnicolide D as an active ingredient.

Another object of the present invention is to provide a quasi-drug composition for preventing, improving, or treating lipid-related metabolic diseases containing patulin, dianemycin, or arnicolide D as an active ingredient.

Another object of the present invention is to provide a feed composition for improving lipid metabolism or preventing lipid-related metabolic diseases containing patulin, dianemycin or arnicolide D as an active ingredient.

This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Additionally, the scope of the present application cannot be considered limited by the specific description described below.

In one aspect for achieving the above object, the present invention provides a food composition for improving lipid metabolism or preventing lipid-related metabolic diseases containing patulin, dianemycin or arnicolide D as an active ingredient.

In the present invention, 'patulin (4-hydroxy-4 H -furo[3,2-c]pyran-2(6 H )-one)' refers to a natural compound having the chemical structure of Formula 1 below.

In the present invention, 'dianemycin, (E)-8-[7-hydroxy-2-[2-[6-hydroxy-6-(hydroxymethyl)-3,5-dimethyloxan-2-yl]-9- (5-methoxy-6-methyloxan-2-yl)oxy-4,10-dimethyl-1,6-dioxaspiro[4.5]decan-7-yl]-2,8-dimethyl-1,10-dioxaspiro[4.5] 'decan-9-yl]-2,4,6-trimethyl-5-oxonon-6-enoic acid)' refers to a natural compound having the chemical structure of Formula 2 below.

In the present invention, 'Arnicolide D, [(1S,3aR,5R,5aR,8aR,9S,9aR)-1,5,8a-trimethyl-2,8-dioxo-3a,4,5,5a ,9,9a-hexahydro-1H-azuleno[6,5-b]furan-9-yl] 2-methylprop-2-enoate' refers to a natural compound having the chemical structure of Formula 3 below.

In the present invention, patulin, dianemycin, and arnicolide D can be obtained from biological sources known in the art, or chemically synthesized or sold.

In the present invention, patulin, dianemycin or arnicolide D includes pharmaceutically or foodologically acceptable salt forms within the range having the same efficacy. These salts are pharmaceutically or foodologically acceptable salts and may be basic salts or acid salts. The basic salts may be used in the form of either organic base salts or inorganic base salts, such as sodium salts, potassium salts, or calcium salts. , lithium salt, magnesium salt, cesium salt, aminium salt, ammonium salt, triethylaminium salt, and pyridinium salt.

A useful type of such salt is an acid salt, an acid addition salt formed by a free acid. Inorganic acids and organic acids can be used as free acids. Hydrochloric acid, hydrobromic acid, sulfuric acid, sulfurous acid, phosphoric acid, double phosphoric acid, and nitric acid can be used as inorganic acids, and citric acid, acetic acid, maleic acid, malic acid, and fumaric acid can be used as organic acids. , glucolic acid, methanesulfonic acid, benzenesulfonic acid, camphorsulfonic acid, oxalic acid, malonic acid, glutaric acid, acetic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, Glutamic acid, citric acid, aspartic acid, stearic acid, etc. may be used, but are not limited thereto and may include all salts formed using various inorganic acids and organic acids commonly used in the art.

In the present invention, “lipid” is a general term for substances that are soluble in organic solvents among the components of living things, such as triglycerides, cholesterol, phospholipids, free fatty acids, or the like. It can be a combination.

In the present invention, “improvement of lipid metabolism” may include inhibiting intracellular lipid accumulation, improving blood lipids, reducing body weight, reducing body fat, improving obesity or blood cholesterol, etc.

In the present invention, "histone acetyltransferase (HAT)" is involved in the acetylation of histones, and histone acetyltransferase increases the acetylation of hormone receptors, causing excessive expression of each target protein. It is also involved in increasing the growth and development of cancer (Biochem. Pharmacol. (2005) 69(8): 1205-1213), and hyperacetylation of histones can cause diseases such as obesity, diabetes, Alzheimer's disease, Parkinson's disease, and epilepsy. It has been reported to be related to degenerative brain disease. C/EBP (CCAAT/Enhancer-binding Protein), a factor related to obesity, is known to stimulate and activate PPAR-α/γ when hyperacetylation occurs due to HAT protein, thereby promoting differentiation and development of adipocytes and causing obesity. There is (Melina, M. M. et al. Biochem. Cell Biol. 85:397-410, 2007). In addition, it inhibits the accumulation of neutral fat in adipocytes by suppressing HAT activity and reducing the expression of genes related to fat synthesis, and a study on a material for preventing and treating simple fatty liver disease has been reported (J Korean Soc Food Sci Nutr 46 ( 2), 169~176 (2017)).

For example, the composition of the present invention improves blood lipid concentration and has the effect of preventing, improving or treating lipid-related diseases such as obesity by inhibiting the activity of histone acetyltransferase and suppressing lipid accumulation.

The composition of the present invention is not limited thereto, but may be implemented in the form of various compositions for external use, such as food (including food additive) compositions, pharmaceutical compositions, quasi-drug compositions, and feed (feed additive) compositions.

The food composition of the present invention includes all forms of functional food, nutritional supplements, health food, food additives, and feed, and includes animals including humans and livestock. Targeted for consumption. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

Food compositions of this type can be prepared in various forms according to conventional methods known in the art. General foods include, but are not limited to, beverages (including alcoholic beverages), fruit and its processed foods (canned fruit, bottled food, jam, marmalade, etc.), fish, meat, and their processed foods (ham, sausage, corned beef, etc.) , bread and noodles (udon, buckwheat noodles, ramen, spagate, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (butter, cheese, etc.), edible plant oils, margarine, vegetable protein, retort food, frozen food. , can be manufactured by adding the above active ingredients to various seasonings (soybean paste, soy sauce, sauce, etc.).

Additionally, nutritional supplements can be manufactured by adding the above-mentioned active ingredients to capsules, tablets, pills, etc. In addition, health functional foods are not limited to this, but for example, they can be manufactured in the form of tea, juice, and drinks and consumed by liquefying, granulating, encapsulating, and powdering so that they can be consumed as health drinks. Additionally, in order to use the patulin, dianemycin or arnicolide D in the form of a food additive, it can be prepared and used in the form of powder or concentrate. Additionally, it can be prepared in the form of a composition by mixing it with known active ingredients known to be effective in improving lipid metabolism.

In addition to the above, the health food of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, It may contain glycerin, alcohol, or carbonating agent.

In one aspect for achieving the above object, the present invention provides a pharmaceutical composition for preventing, improving or treating lipid-related metabolic diseases containing patulin, dianemycin or arnicolide D as an active ingredient.

In the present invention, “lipid-related metabolic disease” is a term referring to diseases that may occur due to lipid accumulation, such as obesity, diabetes, hypertension, hypercholesterolemia, hyperlipidemia, hyperinsulinemia, arteriosclerosis, stroke, dyslipidemia, or fatty liver. etc. can be mentioned.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc.

Carriers for parenteral administration may also include water, suitable oils, saline solutions, aqueous glucose and glycols, and the like. Additionally, stabilizers and preservatives may be additionally included. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.

The pharmaceutical composition of the present invention can be administered to mammals, including humans, by any method. For example, it can be administered orally or parenterally, and the parenteral administration method is not limited to this, but is intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, and intraperitoneal. , may be administered intranasally, enterally, topically, sublingually, or rectally.

The pharmaceutical composition of the present invention can be formulated into a preparation for oral administration or parenteral administration according to the administration route described above. When formulated, one or more buffers (e.g. saline or phosphate buffered saline (PBS)), antioxidants, bacteriostatic agents, chelating agents (e.g. EDTA or glutathione), fillers, extenders, binders, adjuvants (e.g. For example, aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrants or surfactants, diluents or excipients.

Solid preparations for oral administration include tablets, pills, powders, granules, solutions, gels, syrups, slurries, suspensions or capsules, etc. These solid preparations include the pharmaceutical composition of the present invention and at least one excipient, e.g. For example, starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol and maltitol. It can be prepared by mixing cellulose, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropylmethyl-cellulose, or gelatin. For example, tablets or dragees can be obtained by combining the active ingredient with solid excipients, grinding them, adding suitable auxiliaries, and processing them into a granule mixture.

In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, or syrups. In addition to the commonly used simple diluents such as water or liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, or preservatives may be included. there is. In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant, and anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives may be additionally included. .

When administered parenterally, the pharmaceutical composition of the present invention can be formulated with a suitable parenteral carrier in the form of injections, transdermal administration, and nasal inhalation according to methods known in the art. The above injections must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. For injections, examples of suitable carriers include, but are not limited to, solvents or dispersion media including water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), mixtures thereof, and/or vegetable oils. You can. More preferably, suitable carriers include Hanks' solution, Ringer's solution, PBS containing triethanolamine, or sterile water for injection, and isotonic solutions such as 10% ethanol, 40% propylene glycol, and 5% dextrose. . In order to protect the injection from microbial contamination, it may additionally contain various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. Additionally, in most cases, the injection may additionally contain an isotonic agent such as sugar or sodium chloride.

In the case of transdermal administration, forms such as ointments, creams, lotions, gels, external solutions, paste preparations, linear preparations, and aerol preparations are included. Here, 'transdermal administration' means administering a pharmaceutical composition topically to the skin so that an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin.

For inhalation administration, the compositions used according to the invention may be packaged in pressurized packs or using a suitable propellant, such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It can be conveniently delivered in the form of an aerosol spray from a nebulizer. For pressurized aerosols, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in inhalers or insufflators can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch. Formulations for parenteral administration are described in Remington's Pharmaceutical Science, 15th Edition, 1975 Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a text generally known in all pharmaceutical chemistry.

The pharmaceutical composition of the present invention can be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, or methods using biological response modifiers.

In the present invention, “prevention” refers to any act of suppressing or delaying the symptoms of a lipid-related metabolic disease by administering the composition of the present invention, and in the present invention, “improvement” refers to the symptoms of a lipid-related metabolic disease by applying the composition of the present invention. This means that it has a mitigating effect. In the present invention, “treatment” refers to any action in which the symptoms of a lipid-related metabolic disease are improved or beneficially changed by administration of the composition of the present invention.

In one aspect for achieving the above object, the present invention provides a quasi-drug composition for preventing, improving or treating lipid-related metabolic diseases containing patulin, dianemycin or arnicolide D as an active ingredient.

As used in the present invention, the term "quasi-drug" refers to articles used for the purpose of diagnosing, treating, mitigating, treating, or preventing diseases in humans or animals that are not instruments, machines, or devices, and that have pharmacological effects on the structure and function of humans or animals. Among the products used for the purpose of influencing, it refers to products excluding those that are not instruments, machines, or devices. One specific example may include preparations for internal use, but is not limited thereto, and the formulation method, dosage, and use method of quasi-drugs , components, etc. can be appropriately selected from conventional techniques known in the technical field.

In addition to the above ingredients, the quasi-drug composition of the present invention may further include pharmaceutically acceptable carriers, excipients, or diluents as needed. The pharmaceutically acceptable carrier, excipient, or diluent is not limited as long as it does not impair the effect of the present invention, and includes, for example, fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, sweeteners, fragrances, preservatives, etc. It can be included.

In one aspect for achieving the above object, the present invention provides a feed composition for improving lipid metabolism or preventing lipid-related metabolic diseases containing patulin, dianemycin, or arnicolide D as an active ingredient.

In the present invention, the feed composition may be formulated in the form of a normal feed and may include known feed ingredients. It can also be used as a feed additive, which is added to the feed used in the form of an additive. The feed additive of the present invention corresponds to supplementary feed under the Feed Management Act, and includes mineral preparations such as sodium bicarbonate (sodium bicarbonate), bentonite, magnesium oxide, and complex minerals, and trace minerals such as zinc, copper, cobalt, and selenium. Preparations, vitamin preparations such as kerotene, vitamins A, D, E, nicotinic acid, and vitamin B complex, protective amino acid preparations such as methionine and lysic acid, protective fatty acid preparations such as fatty acid calcium salts, probiotics (lactic acid bacteria), yeast cultures, molds Live bacteria such as fermented products, yeast agents, etc. may be additionally included.

The feed or feed additive of the present invention can be applied to a number of animal diets, including mammals, poultry, and fish.

Patulin, dianemycin or arnicolide D of the present invention improves blood lipid concentration by suppressing lipid accumulation and has the effect of preventing, improving or treating lipid-related metabolic diseases, and is used as a material for food, medicine, etc. possible.

Figure 1 shows the results of suppressing lipid accumulation when treated with patulin.
Figure 2 shows the results of suppressing lipid accumulation when treated with dianemycin.
Figure 3 shows the results of suppressing lipid accumulation when treated with arnicolide D.
Figure 4 shows the results of inhibition of histone acetyltransferase (HAT) activity when treated with patulin.
Figure 5 shows the results of inhibition of histone acetyltransferase (HAT) activity when treated with dianemycin.
Figure 6 shows the results of inhibition of histone acetyltransferase (HAT) activity when treated with arnicolide D.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1: Confirmation of lipid accumulation inhibition effect of Patulin

To confirm the effect of patulin on suppressing lipid accumulation, a mouse normal liver cell line (AML12) was treated with OPA (oleic acid, palmitic acid) complex to induce lipid accumulation.

AML12 cells, a normal mouse liver cell line, were treated with Dulbecco's modified cell culture medium containing 10% Fetal Bovine Serum (FBS, WelGene Biopharmaceuticals, Korea), Insulin-Transferrin-Selenium (ITS-G, Gibco, USA), and Penicillin-Streptomycin (WelGene Biopharmaceuticals, Korea). Eagle's medium (DMEM, WelGene Biopharmaceuticals, Korea) was added and cultured under conditions of 5% CO ₂ and 37°C. AML12 cells were distributed in a 24 well plate at a number of 5x10 ⁴ /well, and after 24 hours, the normal control group was treated with Bovine Serum Albumin (BSA), and the test control group was treated with oleic acid and palmitic acid to induce lipid accumulation. ) complex (OPA) was treated, and the test group was treated with 20 μM of OPA and patulin. Oil Red O (ORO) staining was performed to confirm the degree of intracellular lipid accumulation in AML12 cells. When lipid accumulation was complete in the OPA-treated group, the medium was removed, the cells were washed with phosphate buffered saline (PBS), and then treated with 3.7% formalin and fixed for 1 hour at room temperature. Formalin was completely removed, rinsed with 60% isopropanol, Oil Red O working solution was added and dyed for over 30 minutes. At this time, the Oil Red O working solution was diluted in distilled water at a ratio of 6:4 and then filtered. After staining, the Oil Red O working solution was completely removed, washed with distilled water, and the staining state of the cells was checked under a microscope. To measure absorbance, the cells were eluted with 100% isopropanol and measured at a wavelength of 506 nm using a Microplate reader (Molecular Devices, THERMOmax). The optical density (OD) was measured. The measured OD value of the test control group was set as 100%, and the degree of lipid accumulation inhibition of the test group (patulin group) was expressed relative to the test control group (OPA group).

When treated with 20 μM patulin, it was confirmed that lipid accumulation was significantly suppressed compared to the OPA-treated group (p < 0.05) (Figure 1).

Example 2: Confirmation of lipid accumulation inhibition effect of Dianemycin

To confirm the effect of dianemycin on suppressing lipid accumulation, a mouse normal liver cell line (AML12) was treated with OPA (oleic acid, palmitic acid) complex to induce lipid accumulation.

AML12 cells, a normal mouse liver cell line, were treated with Dulbecco's modified cell culture medium containing 10% Fetal Bovine Serum (FBS, WelGene Biopharmaceuticals, Korea), Insulin-Transferrin-Selenium (ITS-G, Gibco, USA), and Penicillin-Streptomycin (WelGene Biopharmaceuticals, Korea). Eagle's medium (DMEM, WelGene Biopharmaceuticals, Korea) was added and cultured under conditions of 5% CO ₂ and 37°C. AML12 cells were distributed in a 24 well plate at a number of 5x10 ⁴ /well, and after 24 hours, the normal control group was treated with Bovine Serum Albumin (BSA), and the test control group was treated with oleic acid and palmitic acid to induce lipid accumulation. ) complex (OPA) was treated, and the test group was treated with 20 μM of OPA and dianemycin. Oil Red O (ORO) staining was performed to confirm the degree of intracellular lipid accumulation in AML12 cells. When lipid accumulation was complete in the OPA-treated group, the medium was removed, the cells were washed with phosphate buffered saline (PBS), and then treated with 3.7% formalin and fixed for 1 hour at room temperature. Formalin was completely removed, rinsed with 60% isopropanol, Oil Red O working solution was added and dyed for over 30 minutes. At this time, the Oil Red O working solution was diluted in distilled water at a ratio of 6:4 and then filtered. After staining, the Oil Red O working solution was completely removed, washed with distilled water, and the staining state of the cells was checked under a microscope. To measure absorbance, the cells were eluted with 100% isopropanol and measured at a wavelength of 506 nm using a Microplate reader (Molecular Devices, THERMOmax). The optical density (OD) was measured. The measured OD value of the test control group was set as 100%, and the degree of lipid accumulation inhibition of the test group (dianemycin group) was expressed relative to the test control group (OPA group).

It was confirmed that when dianemycin was treated at 20 μM, lipid accumulation was significantly suppressed compared to the OPA-treated group (p < 0.05) (Figure 2).

Example 3: Confirmation of lipid accumulation inhibition effect of Arnicolide D

To confirm the effect of arnicolide D on suppressing lipid accumulation, a mouse normal liver cell line (AML12) was treated with OPA (oleic acid, palmitic acid) complex to induce lipid accumulation.

AML12 cells, a normal mouse liver cell line, were treated with Dulbecco's modified cell culture medium containing 10% Fetal Bovine Serum (FBS, WelGene Biopharmaceuticals, Korea), Insulin-Transferrin-Selenium (ITS-G, Gibco, USA), and Penicillin-Streptomycin (WelGene Biopharmaceuticals, Korea). Eagle's medium (DMEM, WelGene Biopharmaceuticals, Korea) was added and cultured under conditions of 5% CO ₂ and 37°C. AML12 cells were distributed in a 24 well plate at a number of 5x10 ⁴ /well, and after 24 hours, the normal control group was treated with Bovine Serum Albumin (BSA), and the test control group was treated with oleic acid and palmitic acid to induce lipid accumulation. ) complex (OPA) was treated, and the test group was treated with OPA and 20 μM of arnicolide D. Oil Red O (ORO) staining was performed to confirm the degree of intracellular lipid accumulation in AML12 cells. When lipid accumulation was complete in the OPA-treated group, the medium was removed, the cells were washed with phosphate buffered saline (PBS), and then treated with 3.7% formalin and fixed for 1 hour at room temperature. Formalin was completely removed, rinsed with 60% isopropanol, Oil Red O working solution was added and dyed for over 30 minutes. At this time, the Oil Red O working solution was diluted in distilled water at a ratio of 6:4 and then filtered. After staining, the Oil Red O working solution was completely removed, washed with distilled water, and the staining state of the cells was checked under a microscope. To measure absorbance, the cells were eluted with 100% isopropanol and measured at a wavelength of 506 nm using a Microplate reader (Molecular Devices, THERMOmax). The optical density (OD) was measured. The measured OD value of the test control group was set as 100%, and the degree of lipid accumulation inhibition of the test group (Arnicolide D group) was expressed relative to the test control group (OPA group).

When treated with 20 μM of arnicolide D, it was confirmed that lipid accumulation was significantly suppressed compared to the OPA-treated group (p < 0.05) (Figure 3).

Example 4: Confirmation of the inhibitory effect of patulin on histone acetyltransferase (HAT) activity

To confirm the inhibitory effect of patulin on histone acetyltransferase activity, the activity of histone acetyltransferase was measured in vitro using a HAT (histone acetyltransferase) activity kit.

After adding 20 μg of nuclear extract (NE; 4 ul) and 20 μM patulin (2 μl) (equivalent amount of DMSO for 0 μg/ml) to a 96 well plate, the final volume was 40 μl. Distilled water (DW) was added as much as possible. For background discrimination, a well containing only 40 μl of DW without NE or materials was prepared. After mixing the commercially available substrate histone H3 tail (5 μl), cofactor acetyl-CoA (5 μl), and NADH generating enzyme (8 μl) in 2 68 μl was dispensed into each well. The OD value was measured at a wavelength of 440 nm using a microplate reader (Molecular Devices, THERMOmax), and this was regarded as T0 HAT activity. After incubation at 37°C for 60 minutes, the OD value was remeasured at the same wavelength and was called T60 HAT activity. In order to calculate the final changed HAT activity, the background was corrected by subtracting the T0 and T60 backgorund OD values from the HAT activity OD values of T0 and T60, and the HAT activity at Δ60 minutes (OD value of T60 - OD value of T0) was calculated. After that, the activity of the positive control reaction well showing only HAT activity of NE was set to 1 to relatively indicate the degree of concentration-dependent inhibition of HAT activity by patulin.

When treated with 20 μM patulin, it was confirmed that HAT activity was significantly inhibited compared to nuclear extract (NE) (p<0.05) (Figure 4).

Example 5: Confirmation of the inhibitory effect of dianemycin on histone acetyltransferase (HAT) activity

To confirm the effect of dianemycin on inhibiting histone acetyltransferase activity, the activity of histone acetyltransferase was measured in vitro using a HAT (histone acetyltransferase) activity kit.

After adding 20 μg of nuclear extract (NE; 4 ul) and 20 μM dianemycin (2 μl) (equivalent amount of DMSO for 0 μg/ml) to a 96 well plate, the final volume was 40 μl. Distilled water (DW) was added to make it . For background discrimination, a well containing only 40 μl of DW without NE or materials was prepared. After mixing the commercially available substrate histone H3 tail (5 μl), cofactor acetyl-CoA (5 μl), and NADH generating enzyme (8 μl) in 2 68 μl was dispensed into each well. The OD value was measured at a wavelength of 440 nm using a microplate reader (Molecular Devices, THERMOmax), and this was regarded as T0 HAT activity. After incubation at 37°C for 60 minutes, the OD value was remeasured at the same wavelength and was called T60 HAT activity. In order to calculate the final changed HAT activity, the background was corrected by subtracting the T0 and T60 backgorund OD values from the HAT activity OD values of T0 and T60, and the HAT activity at Δ60 minutes (OD value of T60 - OD value of T0) was calculated. Afterwards, the activity of the positive control reaction well showing only the HAT activity of NE was set to 1 to relatively indicate the degree of concentration-dependent inhibition of HAT activity by dianemycin.

When dianemycin was treated at 20 μM, HAT activity was confirmed to be significantly inhibited compared to nuclear extract (NE) (p<0.05) (FIG. 5).

Example 6: Confirmation of the inhibitory effect of arnicolide D on histone acetyltransferase (HAT) activity

To confirm the inhibitory effect of arnicolide D on histone acetyltransferase activity, the activity of histone acetyltransferase was measured in vitro using a HAT (histone acetyltransferase) activity kit.

After adding 20 μg of nuclear extract (NE; 4 ul) and 20 μM arnicolide D (2 μl) (equivalent amount of DMSO for 0 μg/ml) to a 96 well plate, the final volume was 40. Distilled water (DW) was added to make up to μl. For background discrimination, a well containing only 40 μl of DW without NE or materials was prepared. After mixing the commercially available substrate histone H3 tail (5 μl), cofactor acetyl-CoA (5 μl), and NADH generating enzyme (8 μl) in 2 68 μl was dispensed into each well. The OD value was measured at a wavelength of 440 nm using a microplate reader (Molecular Devices, THERMOmax), and this was regarded as T0 HAT activity. After incubation at 37 °C for 60 minutes, the OD value was remeasured at the same wavelength and was called T60 HAT activity. In order to calculate the final changed HAT activity, the background was corrected by subtracting the T0 and T60 backgorund OD values from the HAT activity OD values of T0 and T60, and the HAT activity at Δ60 minutes (OD value of T60 - OD value of T0) was calculated. Afterwards, the activity of the positive control reaction well showing only HAT activity of NE was set to 1 to relatively indicate the degree of concentration-dependent inhibition of HAT activity by arnicolide D.

When treated with 20 μM arnicolide D, it was confirmed that HAT activity was significantly inhibited compared to nuclear extract (NE) (p<0.05) (FIG. 6).

Claims (13)

A food composition for improving lipid metabolism or preventing lipid-related metabolic diseases, comprising patulin, dianemycin, or arnicolide D as an active ingredient. According to paragraph 1,
A food composition wherein the improvement of lipid metabolism includes inhibiting intracellular lipid accumulation, improving blood lipids, reducing body fat, or reducing body weight. According to paragraph 2,
The composition is a food composition for inhibiting histone acetyltransferase (HAT) activity. A health functional food for improving lipid metabolism or preventing lipid-related metabolic diseases, comprising the food composition according to claims 1 to 3. According to paragraph 4,
A health functional food wherein the improvement of lipid metabolism includes inhibiting intracellular lipid accumulation, improving blood lipids, reducing body fat, or improving blood cholesterol. According to paragraph 4,
The health functional food is one or more dosage forms selected from the group consisting of health functional food preparations such as tablets, capsules, pills, granules, liquid, powder, flakes, paste, syrup, gel, jelly, bars, beverages, gums, and candies. It is a health functional food. A pharmaceutical composition for preventing, improving or treating lipid-related metabolic diseases containing patulin, dianemycin or arnicolide D as an active ingredient. In clause 7,
A pharmaceutical composition, wherein the lipid-related metabolic disease is one or more selected from the group consisting of obesity, diabetes, hypertension, hypercholesterolemia, hyperlipidemia, hyperinsulinemia, arteriosclerosis, stroke, dyslipidemia, and fatty liver. In clause 7,
The composition is a pharmaceutical composition for inhibiting histone acetyltransferase (HAT) activity. A quasi-drug composition for preventing, improving, or treating lipid-related metabolic diseases containing patulin, dianemycin, or arnicolide D as an active ingredient. According to clause 10,
The lipid-related metabolic disease is at least one selected from the group consisting of obesity, diabetes, hypertension, hypercholesterolemia, hyperlipidemia, hyperinsulinemia, arteriosclerosis, stroke, dyslipidemia, and fatty liver. According to clause 10,
The composition is a quasi-drug composition for inhibiting histone acetyltransferase (HAT) activity. A feed composition for improving lipid metabolism or preventing lipid-related metabolic diseases, comprising patulin, dianemycin, or arnicolide D as an active ingredient.