WO2014208799A1 - Composition for preventing or treating fatty liver comprising evodiae fructus extract and alkaloid compound isolated therefrom as active ingredients - Google Patents

Abstract

The present invention relates to a composition for preventing or treating fatty liver, the composition comprising Evodiae fructus extract and an alkaloid compound isolated therefrom as active ingredients. The Evodiae fructus extract and alkaloid compound isolated therefrom of the present invention are stable inside the body, increase the activity of ANT1 enzyme, and promote lipid oxidation in liver tissue in a fatty-liver animal model and thereby effectively suppress lipid accumulation and triglyceride production, and thus can be used to advantage in the prevention or treatment of fatty liver and in particular non-alcoholic fatty liver.

Description

Composition for the prevention or treatment of fatty liver, comprising sesame oil extract and alkaloid compound isolated therefrom as an active ingredient

The present invention relates to a composition for preventing or treating fatty liver containing Evodia rutaecarpa extract and an alkaloid compound isolated therefrom as an active ingredient.

   Recently, with the increase of the obese population due to the westernization of diet, nonalcoholic fatty liver disease (NAFLD) caused by metabolic syndrome is increasing rapidly. Non-alcoholic fatty liver disease is a disease that has no history of alcohol consumption, but has similar tissue findings as alcoholic fatty liver. Obesity is known as the most important cause. Currently, the prevalence of non-alcoholic fatty liver is the most common cause of liver dysfunction. The most important mechanism of the development of non-alcoholic fatty liver disease is insulin resistance, which is considered to be a common etiology of metabolic syndrome such as diabetes, obesity and hyperlipidemia. Each disease belonging to the metabolic syndrome may be correlated or independently affect the progression, severity and complications of NAFLD. Some of the NAFLDs come in the form of nonalcoholic steatohepatitis (NASH), which is accompanied by the destruction of hepatocytes, and some progress to liver cirrhosis or liver cancer through liver fibrosis.

Fatty liver disease caused by metabolic syndrome (1) increases the amount of free fatty acids from adipocytes into the liver, resulting in fat accumulation, and (2) an important role in the biosynthesis of triglycerides due to hyperglycemia and hyperinsulinemia. It is known to activate SREBP-1c, a transcription factor that causes fat accumulation in liver tissue. Recently, it has been reported that metformin and PPAR-ligand, which activate AMP-activated protein kinase (AMPK), which are involved in cellular energy metabolism, can prevent the development of fatty liver by inhibiting SREBP-1c activity. It's controversial. In addition, there are no reports of drugs that can efficiently regulate SREBP-1c other than metformin and PPAR-ligand. Recently, an antioxidant such as vitamin E and ursodeoxycholic acid (Ursa), which is a hepatocellular protective agent, have been used for the treatment of nonalcoholic fatty liver disease, but there is no clinically significant therapeutic effect. Therefore, the treatment of non-alcoholic fatty liver disease using these drugs is currently not clinically recommended, and only basic treatments such as weight loss, diet therapy and exercise therapy are recommended. to be. Therefore, if the development of a technology that can effectively treat the disease is considered to have a great economic and social ramifications.

On the other hand, Evodia rutaecarpa is a deciduous shrub that grows in the southern part of Korea. In oriental medicine, it is called sewage or sewage oil which is obtained by picking green and brown immature fruit around September. It is used for dry, dry, detoxification and diuretic. Sewage extract has been reported to have analgesic activity in experimental animals and is known to increase blood flow improvement. Other efficacies in filthy feeding are known to be effective in treating abdominal pain, headache pain, diarrhea, allodynia and hypertension, and are known to be effective in removing rashes and eczema caused by the skin on the head. Patents and documents related to such sewage oil "whitening cosmetic composition containing sewage oil extract (Korean Patent No. 10-345225)", "cholesterol esterase inhibitor composition containing sewage extract (Korea Patent No. 10-399529) However, the prevention and treatment effect of fatty liver of alkaloid compounds isolated from sesame oil extract and sesame oil extract is not known.

In general, it is highly likely that new active ingredients can be found from natural medicines used in traditional medicine, rather than efforts by experimental modifications of existing drugs. Since it has been used for a long time, there is an advantage that there is little concern about toxicity by drugs.

Therefore, the present inventors searched about 1500 kinds of natural products to develop new non-alcoholic fatty liver preventive or therapeutic substances with low toxicity from natural products, and separated five kinds of alkaloid compounds as active substances from sewage extract. The present invention was completed by confirming that the isolated compound has a prophylactic or therapeutic effect on non-alcoholic fatty liver.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating fatty liver, comprising sesame oil extract and alkaloid compound isolated therefrom as an active ingredient.

Another object of the present invention to provide a food composition for preventing or improving fatty liver comprising sewage oil extract and alkaloid compound isolated therefrom as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of fatty liver comprising sewage oil extract and alkaloid compounds isolated therefrom as an active ingredient.

In another aspect, the present invention provides a food composition for preventing or improving fatty liver comprising a sewage oil extract and an alkaloid compound separated therefrom as an active ingredient.

The present invention relates to a composition for the prevention or treatment of fatty liver, comprising sesame oil extract and alkaloid compounds isolated therefrom as an active ingredient. The sewage extract of the present invention and the alkaloid compound isolated therefrom have in vivo stability, increase the activity of ANT1 enzyme, promote fatty acidization in liver tissue of fatty liver animal model and thereby produce fat accumulation and triglyceride By effectively inhibiting, it can be usefully used for the prevention or treatment of fatty liver, especially non-alcoholic fatty liver.

1 is a diagram showing the HPLC analysis spectrum of the sewage ethanol extract.

Figure 2 is a diagram showing the ANT1 activity of alkaloid compounds isolated from the sesame oil extract.

Figure 3 is a diagram showing the effect of inhibiting fat accumulation in liver tissue of the evodiamine of db / db mice.

Figure 4 is a diagram showing the effect of triglycerides in the liver tissue of the evodiamine of db / db mice.

Fig. 5 is a diagram showing the effect of inhibiting fat accumulation in liver tissue of evodiamine in a fatty liver animal model by LXR antagonist.

6 is a diagram showing the effect of inhibiting triglyceride production in the liver tissue of the evodiamine of a fatty liver animal model by LXR antagonist.

7 is a diagram showing the effect of evodiamine on fatty acid oxidation inhibitory action by LXR antagonist treatment.

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

The present invention provides a composition for the prevention or treatment of fatty liver, comprising sesame oil extract and alkaloid compounds isolated therefrom as an active ingredient.

The composition comprises a pharmaceutical composition or a food composition.

Fructus extract of the present invention can be obtained by the following method. Sewage oil is washed with water to remove debris, dried in the shade and crushed. Sewage oil can be used without limitation, cultivated or commercially available. Appropriate amount of solvent is added to the ground sewage oil powder to allow complete immersion to obtain the sewage oil extract. The extraction method can be impregnated or warmed at room temperature. The extraction solvent is not limited thereto, but may be one or more solvents selected from water, alcohols having 1 to 4 carbon atoms, and mixed solvents thereof, preferably methanol or ethanol, and more preferably ethanol. After filtering and concentrating the sewage oil extract, the sewage oil extract concentrate was suspended in distilled water and fractionated sequentially with hexane, ethyl acetate, butanol and water to obtain a fraction. The ethyl acetate fraction of the fraction showed the best ANT1 activity.

The active material was separated from the sewage oil ethyl acetate fraction having the highest ANT1 activity using liquid chromatography, and the active material showed the best ANT1 activity among the alkaloid compounds.

The alkaloid compound includes evodiamine, evodiamine, rutaecarpine, 7-hydroxyrutaecarpine, norketoyobyrine, and ketoyobyrine. .

The alkaloid compound isolated from the sewage oil extract may be obtained by the following method. Specifically, a known method used for isolation and extraction of plant components, such as extraction with an organic solvent alcohol (methanol, ethanol, propanol, etc.) or a water-soluble alcohol, a distribution of an organic solvent and water, a method by column chromatography, or the like alone or suitably Can be easily obtained.

The chromatography used in the present invention is not limited thereto, but silica gel column chromatography, LH-20 column chromatography, thin layer chromatography, or high performance High performance liqid chromatography and the like can be used.

The sesame oil extract and alkaloid compounds isolated therefrom according to the present invention increase the activity of ANT1 enzyme, promote fatty acidization in liver tissue of fatty liver animal model and thereby effectively inhibit fat accumulation and production of triglycerides.

Therefore, sewage oil extract and alkaloid compound isolated therefrom according to the present invention can be used as a medicine or health functional food useful for the prevention or treatment of fatty liver, especially non-alcoholic fatty liver.

The composition of the present invention may be used alone or in the form of a combination or aggregate with other pharmaceutically active compounds.

The sewage oil extract, which is an active ingredient of the composition of the present invention, and the alkaloid-based compound isolated therefrom may be administered in combination with other known preventive or therapeutic drugs for fatty liver.

The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention or treatment of fatty liver.

The composition of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. It may also be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like in the form of conventional formulations, external preparations, suppositories, and sterile injectable solutions. Suitable formulations known in the art are preferably those disclosed in Remington's Pharmaceutical Science, recently, Mack Publishing Company, Easton PA. Carriers, excipients and diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. In formulating the composition, 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 the solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, lactose, It is prepared by mixing gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, 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.

The dosage of the sewage extract of the present invention and the alkaloid compound isolated therefrom depends on the age, sex, and weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, the route of administration, and the judgment of the prescriber. It may vary. Dosage determination based on these factors is within the level of skill in the art and generally dosages range from 0.1 mg / kg / day to approximately 1,000 mg / kg / day. More preferred dosage is 1 mg / kg / day to 500 mg / kg / 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 compounds of the present invention can be administered to a subject by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections. Since the compound of the present invention has little toxicity and side effects, it is a drug that can be used safely even when taken for a long time for the purpose of prevention.

In the present invention, the health functional food refers to a food having a bioregulatory function, such as prevention and treatment of disease, biodefence, immunity, recovery of the disease, inhibition of aging, and should be harmless to the human body when taken in the long term.

The composition of the present invention may be added to a dietary supplement for the purpose of preventing or improving fatty liver. When the composition of the present invention is used as a food additive, the composition may be added as it is or used with other food or food ingredients, and may be appropriately 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). Generally, in the preparation of food or beverages, the compositions of the present invention are added in an amount of up to 15% by weight, preferably up to 10% by weight relative to the raw materials. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, it 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.

There is no particular limitation on the kind of food. Examples of the food to which the substance may be added include meat, sausage, bread, chocolate, candy, snacks, confectionary, pizza, ramen, other noodles, dairy products including gum, ice cream, various soups, beverages, tea, drink, Alcoholic beverages and vitamin complexes, and the like and include all of the health foods in the conventional sense.

The health beverage composition of the present invention may include various flavors or natural carbohydrates, and the like as an additional ingredient, as in a general beverage. The natural carbohydrates described above may be used as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and natural sweeteners such as dextrin and cyclodextrin, and synthetic sweeteners such as saccharin and aspartame. . The proportion of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 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, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, And carbonating agents used in the carbonated beverage. In addition, the composition of the present invention may include a pulp for the production of natural fruit juice, 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 generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, preferred examples, experimental examples, and preparation examples are provided to help understanding of the present invention. However, the following Examples, Experimental Examples and Preparation Examples are merely provided to more easily understand the present invention, but the contents of the present invention are not limited by Examples, Experimental Examples, and Preparation Examples.

Example 1 Isolation and Purification of ANT1 Active Substance from Fructus Extracts

3 kg of sewage fruit was soaked in 10 L of ethanol and extracted for 1 week at room temperature. The sewage oil ethanol extract was concentrated under reduced pressure, and then suspended in water by adding water to the concentrate (345 g), and fractionated sequentially with n-hexane, ethyl acetate and butanol. ANT1 is a carrier protein present in the inner membrane of mitochondria and plays a role (ATP / ADP translocator) to export ATP produced during oxidative phosphorylation of mitochondria into the cytoplasm. Previous studies have shown that mitochondrial protein ANT1 may be an important pathological mechanism for fatty liver prevention. Therefore, in the present invention, in order to search for candidates for the treatment of fatty liver, substances that regulate the activity of ANT1 were searched.

As a result of confirming the activity of ANT1 for each solvent fraction, it was confirmed that strong activity in the ethyl acetate fraction. 48 g of ethyl acetate fractions were subjected to silica gel column chromatography under a solvent gradient of hexane-acetone 1: 0 to 0: 1 to obtain 6 fractions (fr. 1 to 6). Showed this fraction among fraction 2, 3 and 5 are ANT1 activity, the (carboxylic pin to rutile) of Compound (2) 200mg through the crystallization process from fraction 2, the compound of 1g through the same process from a fraction 31 (EVO diamine ) Was obtained. Reverse phase (RP-18) column chromatography was performed again under the conditions of increasing the fraction 5 from 2: 1 to 5: 1 using MeOH-H ₂ O mixed solvent to obtain five smaller fractions (fr 5.1-5.5). Small fraction Fr5.3 was purified by HPLC using 43% MeCN-H ₂ O [RS Tech Optima Pak C18 column 20 mm × 50 mm; 10 μm particle size 2 ml / min; UV detection: 254 nm], followed by Compound 3 (7-hydroxyrutacarpine, tR = 28.5 min), Compound 5 (ketoyovirin, tR = 51.2 min), Compound 4 (norketoyovirin, tR = 65.0 min) was obtained. (See Figure 1).

 Example 2: Structural Analysis of Compounds Isolated from Fructus Extracts

The structure of five compounds isolated from the sewage oil extract obtained in Example 1 was analyzed. The chemical structure of the compound was measured using an ESI mass spectrometer (Electrospray Ionization mass spectrometer) to determine the molecular weight, and the chemical structure was confirmed by ¹ H and ¹³ C-NMR analysis. The chemical formula and chemical properties of the compounds 1 to 5 are shown below.

Compound 1: Evodiamine

Appearance: yellowish powder;

¹ H NMR (500 MHz, Acetone-d6) δ _H (ppm): 10.32 (1H, br, s, 1-NH), 7.93 (1H, dd, J = 1.5, 7.5, H-19), 7.54 (1H , br, d, J = 7.5, H-9), 7.47 (1H, ddd, J = 1.5, 7.5, 8.0, H-17), 7.41 (1H, br, d, J = 8.0, H-16), 7.41 (1H, br, d, J = 8.0, H-12), 7.14 (1H, br, t, J = 8.0, H-18), 7.08 (1H, br, t, J = 7.5, H-10) , 7.04 (1H, ddd, J = 0.5, 7.5, 8.0, H-11), 6.06 (1H, s, H-3), 4.77, 3.22 (each 1H, m, H-5), 1.96 (2H, m , H-6), 2.80 (3H, s, N-CH ₃ );

¹³ C NMR (125 MHz, Acetone-d6) δ _C (ppm): 165.2 (C-21), 150.8 (C-15), 138.1 (C-13), 135.0 (C-2), 134.0 (C-17 ), 130.8 (C-19), 129.3 (C-10), 125.1 (C-8), 123.1 (C-11), 122.4 (C-18), 120.2 (C-9), 119.3 (C-16) , 113.4 (C-20), 112.5 (C-12), 110.8 (C-7), 70.8 (C-3), 41.3 (C-5), 37.3 (N-CH ₃ ), 20.7 (C-6) .

Compound 2: Rutaecarpine

Appearance: light yellow amorphous powder;

¹ H NMR (600 MHz, CDCl ₃ ) δ _H (ppm): 9.67 (1H, br, s, 1-NH), 8.34 (1H, dd, J = 1.2, 7.8, H-19), 7.70 (1H, ddd, J = 1.8, 1.2, 7.2, H-9), 7.65 (1H, d, J = 7.5, 8.0, H-17), 7.41 (1H, br, d, J = 8.0, H-16), 7.41 (1H, br, d, J = 8.0, H-12), 7.14 (1H, br, t, J = 8.0, H-18), 7.08 (1H, br, t, J = 7.5, H-10), 7.04 (1H, ddd, J = 0.5, 7.5, 8.0, H-11), 6.06 (1H, s, H-3), 4.77, 3.22 (each 1H, m, H-5), 1.96 (2H, m, H-6), 2.80 (3H, s, N-CH ₃ );

¹³ C NMR (150 MHz, CDCl ₃ ) δ _C (ppm): 165.2 (C-21), 150.8 (C-15), 138.1 (C-13), 135.0 (C-2), 134.0 (C-17) , 130.8 (C-19), 129.3 (C-10), 125.1 (C-8), 123.1 (C-11), 122.4 (C-18), 120.2 (C-9), 119.3 (C-16), 113.4 (C-20), 112.5 (C-12), 110.8 (C-7), 70.8 (C-3), 41.3 (C-5), 37.3 (N-CH ₃ ), 20.7 (C-6).

Compound 3: 7-Hydroxyrutaecarpe

Appearance: pale yellow powder;

¹ H NMR (500 MHz, Acetone-d6) δ _H (ppm): 10.32 (1H, br, s, 1-NH), 8.23 (1H, dd, J = 1.5, 8.5, H-19), 7.78 (1H) , ddd, J = 1.5, 8.5, 9.0, H-17), 7.71 (1H, br, d, J = 8.0, H-9), 7.62 (1H, br, d, J = 9.0, H-12), 7.46 (1H, br, t, J = 8.0, H-18), 7.31 (1H, br, t, J = 7.5, H-10), 7.26 (1H, br, d, J = 9.0, H-16) , 7.14 (1H, br, t, J = 7.5, H-11), 6.91 (1H, dd, J = 1.5, 5.0, H-5), 3.55 (1H, dd, J = 1.5, 17.0, H-6eq ), 3.43 (1H, doublet of doublets, J = 5.0, 17.0, H-6ax);

¹³ C NMR (125 MHz, Acetone-d6) δ _C (ppm): 161.7 (C-21), 148.9 (C-3), 145.2 (C-15), 139.9 (C-13), 135.4 (C-17 ), 127.9 (C-19), 127.8 (C-18), 127.5 (C-2), 127.3 (C-8), 126.8 (C-16), 125.8 (C-10), 122.4 (C-20) , 120.9 (C-11), 120.8 (C-9), 115.7 (C-7), 74.8 (C-5), 28.3 (C-6).

Compound 4: Norketoyobyrine

Appearance: pale yellow amorphous powder;

¹ H NMR (500 MHz, Acetone-d6) δ _H (ppm): 10.04 (1H, br, s, 1-NH), 8.16 (1H, dd, J = 1.5, 7.5, H-19), 7.86 (1H , br, d, J = 8.0, H-12), 7.77 (1H, ddd, J = 1.5, 7.5, 8.0, H-17), 7.44 (1H, br, d, J = 8.5, H-16), 7.39 (1H, br, d, J = 8.0, H-18), 7.30 (1H, ddd, J = 0.5, 7.5, 8.0, H-18), 7.24 (1H, s, H-14), 7.11 (1H , ddd, J = 0.5, 7.5, 8.0, H-10), 7.06 (1H, ddd, J = 0.5, 7.5, 8.0, H-11), 4.32, (2H, m, H-5), 3.10 (2H , m, H-6);

¹³ C NMR (125 MHz, Acetone-d6) δ _C (ppm): 162.2 (C-21), 151.7 (C-3), 141.9 (C-2), 137.8 (C-13), 135.8 (C-17 ), 128.9 (C-19), 128.6 (C-8), 123.5 (C-15), 123.4 (C-18), 123.3 (C-12), 122.2 (C-10), 119.7 (C-11) , 119.6 (C-9), 116.6 (C-7), 115.0 (C-16), 112.8 (C-20), 112.2 (C-14), 43.2 (C-5), 24.7 (C-6).

Compound 5: Ketoyobyrine

Appearance: light yellow amorphous powder;

¹ H NMR (500 MHz, Acetone-d6) δ _H (ppm): 9.88 (1H, br, s, 1-NH), 8.17 (1H, br, d, J = 7.5, H-19), 7.77 (1H , br, t, J = 8.0, H-11), 7.45 (1H, br, d, J = 8.0, H-10), 7.39 (1H, dd, J = 2.0, 7.5, H-12), 7.31 ( 1H, br, t, J = 7.5, 8.0, H-18), 7.27 (1H, d, 8.5, H-9), 7.24 (1H, s, H-14), 6.76 (1H, dd, J = 2.0 , 8.5, H-17), 4.32, (2H, m, H-5), 3.84 (3H, s, -OCH ₃ ), 3.10 (2H, m, H-6);

¹³ C NMR (125 MHz, Acetone-d6) δ _C (ppm): 162.3 (C-21), 154.9 (C-16), 151.7 (C-3), 141.9 (C-2), 135.9 (C-11 ), 133.1 (C-13), 129.3 (C-8), 129.1 (C-19), 124.1 (C-7), 124.0 (C-9), 123.5 (C-18), 116.6 (C-20) , 115.1 (C-10), 112.9 (C-12), 112.8 (C-15), 112.5 (C-14), 101.6 (C-17), 55.9 (-OCH ₃ ), 43.0 (C-5), 24.7 (C-6).

Experimental Example 1 Activity Measurement Using ANT1 Promoter

In order to confirm the feasibility of the sewage oil extract prepared in Example 1 and the compounds 1-5 isolated therefrom as a therapeutic agent for fatty liver, the activity of ANT1 was measured.

The activation activity of ANT1 was measured using the ANT1 promoter-luciferase system. The promoter is a sequence that regulates transcription, and the luciferase gene located behind the nucleotide sequence of the promoter increases in expression as the promoter becomes more active. Therefore, the activity of the promoter can be known from the expression level of luciferase. Since ANT1 is a mitochondrial protein, mouse endothelial cell (endothelioma) bEnd.3 cells with relatively high distribution of mitochondria were selected to measure promoter activity. bEnd.3 cells were collected by treatment with 0.25% trypsin, and then the supernatant was removed and suspended in DMEM medium without bovine serum. 3 μl of lipofectamine (lipopectAMINE, life Technologies, Inc) was added to 100 μl of the DMEM, and the mixture was slowly mixed and allowed to stand at room temperature for 15 minutes. Standard to consistently match the efficiency of 50 ng of pANT1 II-Luc or mpANT1-Luc (plasmid with luciferase gene) and 0.1 μg of pCMV-β-galactosidase (transfection, how DNA is introduced into animal cells) Plasmid with β-galactosidase gene], 0.1 μg of ANT1 expression vector, or a microprecision tube containing a vector to be used as a control. It was left for another 15 minutes. The mixture of lipofectamine and DNA-plasmid and b-End.3 cells were slowly mixed and incubated at room temperature for 20 minutes in suspension, then aliquoted in a 60 mm culture vessel and incubated in a CO ₂ incubator for 24 hours. The next day, 10 μg / ml of each of the sewage extracts prepared in Example 1 and 1 μg / ml of each of the compounds separated therefrom were added thereto, followed by further incubation for 24 hours. bEnd.3 cells were washed twice with PBS, 500 μl of extract solution was added to each culture vessel, and the cells were disrupted and centrifuged at 14,000 rpm for 5 minutes. The obtained supernatant was luciferase and β-galactosidase. Used to measure the activity of. Luciferase activity was obtained by supernatant extracted from 100 μl of cells, 100 μl of 20 nM D-luciferin (substrate luminescent using luciferase) and 300 μl of reaction solution (20 mM glycylglycin, 12.5 mM MgSO 4, 3 mM EGTA, 15 mM potassium). phosphate, 1 mM DTT, 1 mM ATP) and measured with a photometer (luminometor, Berthold Clinolumat). In order to correct the efficiency of gene injection, luciferase activity was corrected by measuring the activity of β-galactosidase. The activity of β-galactosidase was 30 μl of cell extract, 66 μl of o-nitrophenyl-β-D-galactoside (4 mg / ml), and 204 μl of reaction solution (0.1 M sodium phosphate, 1 mM MgCl ₂ , 45 mM β). -mercaptoethanol (pH 7.5) was reacted in a 37 ° C thermostat and then absorbance was measured with a spectrophotometer. The results of confirming the activity of ANT1 of the sesame oil extract and alkaloid compounds 1-5 separated therefrom are shown in Table 1 and FIG. 2, respectively.

Table 1

Fraction of Sewage Extract	ethanol	Ethyl acetate	Butanol	water
ANT1 promoter activity of sewage milk (%)	65	76	45	20

As shown in Table 1, it was confirmed that the promoter activity of the ANT1 of the ethyl acetate fraction in the sewage oil extract was the best at 76%.

In addition, as shown in Figure 2, it was confirmed that the average activity of the promoter of ANT1 increased more than two times at a final concentration of 5㎍ / ml of alkaloid compounds 1-5 isolated from sewage oil extract.

Experimental Example 2: Toxicity Experiment

  2-1. Acute Toxicity

      In order to confirm the biostability of the evodiamine (Compound 1) which is a kind of compound isolated from the sesame oil extract, the following experiment was performed.

Twenty ICR mouse strains, which are common mice, were used, and 10 rats were assigned to the control group and 10 rats to the experimental group. The control group was orally administered with only a solvent containing no compound (PEG-400 / tween-80 / EtOH, 8/1/1, v / v / v), and the experimental group was 2.0 g / kg of evodiamine separated from the sesame oil extract. It was administered orally at the concentration. Each mortality was examined 24 hours after administration, and both the control group and the experimental group administered with 2.0g / kg evodiamine survived. It was confirmed that the evodiamine did not show acute toxicity at the concentration of 2.0g / kg.

2-2. Organ and Histotoxicity

The following experiment was conducted to confirm the long-term and histotoxicity of the evodiamine (Compound 1), which is a kind of compound isolated from the sesame oil extract.

OLETF rats were used as experimental animals, and only control solvent was administered orally with evodiamine at a concentration of 200 mg / kg. Four weeks later, blood was collected from the animals, and blood concentrations of GOT (glutamate-oxalate-transferase), GPT (glutamate-pyruvate-transferase), creatine kinase and creatinine were measured. As a result, the experimental group of evodiamine did not show any difference between GOT and GPT, which are known to be related to hepatotoxicity, creatinine, which is an indicator of kidney toxicity, and creatine kinase, which is an indicator of muscle toxicity. In addition, the heart, lung, pancreas, adrenal gland, liver, kidneys and muscles were cut from each animal and histological observations were performed by optical microscopy through conventional tissue fabrication procedures, but specific abnormalities or toxic effects were induced. The results could not be confirmed. The results confirm that the evodiamine is a stable material without long-term and histotoxicity.

Experimental Example 3: Elucidation of fatty liver prevention and treatment effects of evodiamine isolated from sesame oil extract in db / db mice>

In order to determine whether the compound isolated from the sesame oil extract has an effect of preventing fatty liver in db / db mice, the following experiment was performed.

 Although db / db mice are well known as obese and type 2 diabetic animal models, nonalcoholic fatty liver is also well developed and thus used as animal models. Normal 8-week-old mice (lean) were used as a normal group, and water or evodiamine (Compound 1, 200 mg / kg) was orally administered to 8-week-old db / db mice (control group) for 3 weeks.

After 3 weeks, liver tissue of each group of mice was separated and observed, and the results are shown in FIGS. 3 and 4.

As shown in FIG. 3, a marked increase in fat accumulation was observed in liver tissue of normal mice in the normal group. On the other hand, administration of 200 mg / kg of evodiamine significantly reduced fat accumulation in liver tissues of db / db mice.

In addition, as shown in Figure 4, it was confirmed that the triglycerides in the increased liver tissue of db / db mice significantly decreased upon administration of evodiamine.

Through the above results, it can be seen that the evodiamine isolated from the sewage extract has a prophylactic or therapeutic effect of fatty liver in db / db animal model.

<Experiment 4: Determination of fatty liver prevention and treatment effect of evodiamine isolated from sesame oil extract in mice treated with LXR agonist (agonist)>

Liver X receptor (LXR) is a receptor that binds to oxidized cholesterol by-products. LXR activates genes that encode proteins that carry cholesterol from peripheral tissues to the liver when cholesterol by-products increase. In particular, it increases the expression of sterol regulatory factor-binding protein 1c (SREBP1c), the most important enzyme in fatty acid metabolism. Therefore, it is known that treatment with LXR antagonists causes fatty liver.

Male mice of 8-week-old C57BL / 6J were orally administered with water or evodiamine 200 mg / kg for 1 week, with oral administration of 50 mg / kg of LXR agonist. One week later, the liver tissues of the mice in each group were separated and the triglyceride content in the tissues was measured.

The inhibitory effect of evodiamine on fat accumulation and neutral fat production in liver tissues of LXR antagonists in liver tissues are shown in Figs. 5 and 6, respectively, and the effects of evodiamine on fatty acid oxidation inhibition by LXR antagonist treatment. The influence is shown in FIG. 7.

As shown in FIG. 5, it was confirmed that fatty liver production was observed in the treatment of LXR antagonist, and that fatty acid generation induced by LXR antagonist treatment was suppressed when evodiamine was administered together.

In addition, as shown in Figure 6, when the administration of evodiamine it was confirmed that the accumulation of triglycerides in liver tissue significantly.

In addition, as shown in Figure 7, it was confirmed that fatty acid oxidation reduced by the LXR agonist significantly increased upon administration of evodiamine.

Through the above results, it can be seen that the evodiamine isolated from the sesame oil extract has a fatty liver prevention and treatment effect in the animal model inducing fatty liver by treatment with LXR antagonist.

Formulation Example 1: Pharmaceutical Formulation

1-1. Manufacture of tablets

200 g of sesame oil extract and alkaloid compound isolated therefrom

Lactose 175.9g

Potato Starch 180g

32g colloidal silicic acid

Potato Starch 160g

50 g of talc

5 g magnesium stearate

The sewage oil extract and the alkaloid-based compound separated therefrom were mixed with lactose, potato starch and colloidal silicic acid, 10% gelatin solution was added, and then ground and passed through a mesh body. After drying, the mixture obtained by adding potato starch, syrup and magnesium stearate was made into a tablet.

1-2. Preparation of Injection

1 g of sesame oil extract and alkaloid compound isolated therefrom

 0.6 g sodium chloride

 0.1 g of ascorbic acid

 The above composition ratio was dissolved in distilled water to make 100 ml. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

1-3. Manufacture of powder

Sewage Extract and Alkaloid Compound Isolated 20mg

 Lactose 100 mg

 Talc 10 mg

 The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

1-4. Preparation of Capsules

Sewage Extract and Alkaloid Compound Isolated 10mg

 3 mg of crystalline cellulose

 Lactose 14.8 mg

 Magnesium Stearate 0.2 mg

 According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

1-5. Preparation of liquid

Oshuchu extract and alkaloid compound isolated 20mg

 10 g of isomerized sugar

 5 g of mannitol

 Purified water

 According to the conventional method of preparing a liquid solution, each component is added to the purified water to dissolve it, the lemon flavor is appropriately added, the above components are mixed, purified water is added, the whole is purified to 100 ml, and then filled in a brown bottle. The solution is prepared by sterilization.

 Preparation Example 2 Food Preparation Example

2-1. Preparation of Cooking Seasonings

Sewage oil extract and alkaloid-based compounds separated therefrom were prepared for health promotion cooking seasoning at 0.2 to 10 parts by weight, respectively.

2-2. Manufacture of Flour Food

Sewage oil extract and alkaloid-based compounds separated therefrom were added to the flour at 0.1 to 5.0 parts by weight, respectively, and bread, cake, cookies, crackers and noodles were prepared using the mixture to prepare foods for health promotion.

2-3. Preparation of soups and gravy

Sewage oil extract and alkaloid-based compounds separated therefrom were added to the soup and the juice of 0.1 to 1.0 parts by weight, respectively, to prepare meat products for health promotion, soup of noodles and soup.

2-4. Manufacture of dairy products

Sewage oil extract and alkaloid-based compounds separated therefrom were added to the milk at 0.1 to 1.0 parts by weight, respectively, to prepare various dairy products such as butter and ice cream.

2-5. Manufacture of health food

 Sewage Extract and Alkaloid Compound 100mg Isolated from It

 Vitamin Mixture

 70 μg of Vitamin A Acetate

 Vitamin E 1.0 mg

 Vitamin B1 0.13 mg

 Vitamin B2 0.15 mg

 Vitamin B6 0.5 mg

 0.2 μg of vitamin B12

 Vitamin C 10 mg

 Biotin 10 μg

 Nicotinamide 1.7 mg

 Folic acid 50 μg

 Calcium Pantothenate 0.5 mg

 Mineral mixture

 Ferrous Sulfate 1.75 mg

 Zinc Oxide 0.82 mg

 Magnesium carbonate 25.3 mg

 15 mg potassium monophosphate

 Dicalcium Phosphate 55 mg

 Potassium Citrate 90 mg

 Calcium Carbonate 100 mg

 Magnesium chloride 24.8 mg

 Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Preparation Example 3 Beverage Preparation Example

3-1. Vegetable Juice Manufacturing

0.5 g of sesame oil extract and alkaloid compound isolated therefrom

 1,000ml tomato or carrot juice

 The extract was added to juice to prepare vegetable juice for health promotion.

3-2. Fruit juice manufacturing

0.1 g of sesame oil extract and alkaloid compound isolated therefrom

 1,000 ml of apple or grape juice

 The extract was added to juice to prepare fruit juice for health promotion.

 3-3. Manufacture of health drinks

Sewage Extract and Alkaloid Compound 100mg Isolated from It

  15 g of vitamin C

  100 g of vitamin E (powder)

  Iron lactate 19.75 g

  3.5 g of zinc oxide

  Nicotinamide 3.5 g

  0.2 g of vitamin A

  0.25 g of vitamin B1

  0.3 g of vitamin B2

  Water quantification

  After mixing the above components according to the conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 l container, sealed sterilization and refrigerated and then Used to prepare the healthy beverage composition of the invention.

Although the composition ratio is mixed with a component suitable for a favorite beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

Claims (17)

1. A pharmaceutical composition for preventing or treating fatty liver, comprising sesame oil extract as an active ingredient.
2. The pharmaceutical composition for preventing or treating fatty liver according to claim 1, wherein the sewage oil extract is a sewage ethyl acetate fraction.
3. The pharmaceutical composition for preventing or treating fatty liver according to claim 1, wherein the sessile extract extract increases the activity of an adenine nucleotide translocator 1 (ANT1) enzyme.
4. The pharmaceutical composition for preventing or treating fatty liver, according to claim 1, wherein the sesame oil extract promotes fat oxidation in liver tissue.
5. The pharmaceutical composition for preventing or treating fatty liver, according to claim 1, wherein the sewage oil extract inhibits fat accumulation and triglyceride production in liver tissue.
6. The pharmaceutical composition for preventing or treating fatty liver, according to any one of claims 1 to 5, wherein the fatty liver is non-alcoholic fatty liver.
7.  At least one selected from the group consisting of evodiamine, rutaecarpine, 7-hydroxyrutaecarpine, norketoyobyrine and ketoyobyrine Pharmaceutical composition for the prevention or treatment of fatty liver containing the compound as an active ingredient.
8.  8. The pharmaceutical composition for preventing or treating fatty liver of claim 7, wherein the compound is isolated from sesame oil extract.
9.        The pharmaceutical composition for preventing or treating fatty liver of claim 8, wherein the sewage oil extract is separated from the sewage ethyl acetate fraction.
10.  10. The pharmaceutical composition for preventing or treating fatty liver according to any one of claims 7 to 9, wherein the fatty liver is non-alcoholic fatty liver.
11. Food composition for the prevention or improvement of fatty liver comprising a sewage oil extract as an active ingredient.
12. The food composition for preventing or improving fatty liver according to claim 11, wherein the sewage oil extract is an ethyl acetate fraction.
13. The food composition for preventing or improving fatty liver according to claim 11 or 12, wherein the fatty liver is non-alcoholic fatty liver.
14. At least one selected from the group consisting of evodiamine, rutaecarpine, 7-hydroxyrutaecarpine, norketoyobyrine and ketoyobyrine Fatty liver prevention or improvement food composition comprising a compound as an active ingredient.
15. The food composition for preventing or improving fatty liver according to claim 14, wherein the compound is isolated from sewage extract.
16. The food composition for preventing or improving fatty liver according to claim 15, wherein the sewage oil extract is a sewage ethyl acetate fraction.
17. The food composition according to any one of claims 14 to 16, wherein the fatty liver is non-alcoholic fatty liver.

Images