KR101794924B1 - Method for Isolating Isorhamnetin for Prevention or Treatment of Non-alcoholic Fatty Liver Disease derived from Salicornia SPP. - Google Patents

Abstract

The present invention relates to a pharmacological composition for preventing or treating non-alcoholic fatty liver containing Salicornia SPP.-derived isorhamnetin. More specifically, the present invention relates to a pharmacological composition for preventing or treating hepatitis, liver fibrosis, liver cirrhosis, and so on caused by non-alcoholic fatty liver, wherein the pharmacological composition inhibits inflammation reaction in a liver cell and has liver cell protection activity. According to the present invention, Salicornia SPP.-derived isorhamnetin not only inhibits protein expression of iNOS and COX-2, which are representative inflammation markers in liver cells, in a concentration-dependent manner but also increases glutathione synthesis enzymes (GR, GCL) by activating Nrf2, which is a representative cell protection transcription factor which resists oxidation stress. Therefore, the pharmacological composition for preventing or treating non-alcoholic fatty liver containing Salicornia SPP.-derived isorhamnetin can be used to prevent and treat hepatitis, liver fibrosis, liver cirrhosis, and so on which are non-alcoholic fatty liver diseases.

Description

(Isolhamnetin for Prevention or Treatment of Non-Alcoholic Fatty Liver Disease Derived from Salicornia SPP.

The present invention relates to a pharmaceutical composition for the prevention or treatment of nonalcoholic fatty liver disease, which comprises isopramenin derived from a Tochimantim, as an active ingredient. More particularly, the present invention relates to a pharmaceutical composition for preventing or treating nonalcoholic fatty liver disease To a pharmaceutical composition for preventing or treating liver diseases such as hepatitis, hepatic fibrosis and cirrhosis.

According to statistics by the National Statistical Office (NSO) in 2014, cancer, heart disease, and vascular disease, which are the three leading causes of death in Korea, account for almost half of all deaths, accounting for 47.4%, followed by pneumonia, diabetes and liver disease. In particular, 6,635 deaths from liver disease accounted for 13.1% per 100,000 population. Fatty liver is caused by accumulation of excess fat in the liver (mainly triglycerides). Generally, when more than 5% of liver weight is accumulated, it is diagnosed as fatty liver (National Health Information Portal Medical Information). Fatty liver disease is a very common disease so that most (60-80%) of the patients who visited the hospital due to abnormal liver function are fatty liver and 20-30% of the total population is fatty liver.

In addition, 3 out of 10 Korean adults showed fatty liver disease. In 2014, the proportion of patients in the provincial department of the digestive system of Samsung Seoul Hospital was 35.8% in the 30s, 34.3% in the 40s, %, 60 vs 24.6% and 20 vs 24.1%, respectively. The Korean Hepatology Association analyzed information on 750,000 adults who had undergone medical screening from 1988 to 2014, indicating that 70% of diabetic patients, 52% of hypertensive patients, and 38% of metabolic syndrome patients had fatty liver, In addition to the liver-related therapeutic market, the health functional food market is also experiencing a growing interest in consumer-oriented products and consumer needs.

The fatty liver can be divided into non-alcoholic fatty liver disease (NAFLD), which is caused by excessive alcohol intake and alcohol related to obesity due to high fat diet, and 15% of nonalcoholic fat liver fatty liver disease And liver fibrosis, 5% of fatty liver infections are cirrhotic or liver cancer. It is also known that hyperlipidemia, in which the drug overdose and the amount of total cholesterol and triglyceride in the blood vessels are increased to above normal levels, also causes the non-alcoholic fatty liver (NAFLD)

It has been reported that statin drugs such as gemfibrozil and atorvastatin, which are drugs prescribed for the prevention and treatment of nonalcoholic fatty liver disease, cause damage to mitochondria in muscle, It is said to be unsuitable. One of the mechanisms of liver inflammation and hepatic fibrosis, which is a representative disease of nonalcoholic fatty liver, is that active oxygen induced by fatty acid oxidation in hepatocytes activates NFkB, a central transcription factor of inflammation-related signaling pathway, and induces related inflammatory genes, COX-2 and iNOS . ≪ / RTI >

Vitamin E (Tocopherol) and Vitamin C (ascorbic acid) are effective in prevention of fatty liver and hepatic function and histologic examination of patients with hepatitis, and the precursor of glutathione (GSH), an antioxidant naturally present in hepatocytes (N-acetylcysteine, S-adenosyl-methionine [SAM]) is known to be an antioxidant that increases glutathione in the liver and can be used in the treatment of fatty liver. Glutathione (GSH) in the liver is synthesized from γ-glutamylcysteine lyase (GCL), a central synthetic enzyme, and is produced by reduction from oxidized form of GSSG by the action of glutathione reductase (GR) . The increase of glutathione synthetase in hepatocytes such as glutathione reductase (GR) and γ-glutamylcysteine lyase (GCL) has been shown to be involved in cell homeostasis and activation of Nrf2, a typical transcription factor Lt; / RTI > Therefore, it is suggested that antioxidative components derived from natural materials that can inhibit activation of NFkB, an inflammatory factor, and activate cell protective and protective Nrf2 transcription factors may also be effective in the prevention and treatment of fatty liver disease. Therefore, there is a need to develop drugs derived from natural products for the prevention and treatment of new non-alcoholic fatty liver which has no side effects and is inexpensive and has excellent therapeutic effect.

Korean Patent Laid-Open Publication No. 2015-0103847 relates to a composition for preventing or treating a non-alcoholic fatty liver disease containing an extract of Garlic as an active ingredient, wherein the garlic extract is selected from the group consisting of non-alcoholic fatty liver such as fat accumulation, , It is possible to reduce the lipid content of the blood and to inhibit the expression of SREBP-1, a transcription factor controlling fatty acid biosynthesis and PPARγ and C / EBPα, which are adipocyte transcription regulators, , The separation of the active ingredient was not attempted.

Korean Patent No. 0867320 discloses a composition for preventing or treating liver fibrosis (liver cirrhosis) using water extracts of 13 kinds of herb medicines. The composition is used to induce liver fibrosis through a diet supplemented with dimethylnitrosoamine (DMN) Bilirubin, AST, ALT and LDA content of lipid peroxidation in liver tissue were analyzed, but no studies on active ingredients were attempted.

On the other hand, Salicornia europaea ( Salicornia europaea ) is a representative algae plant that is abundant in the coastal waters of the west coast of Korea. It is resistant to salt, and can grow without reduction in growth even in the four-sided cultivation with 200 mM NaCl added. (Obligatory halophyte or True halophyte). In addition to sodium, it also contains minerals such as magnesium, potassium and iron, and a large amount of amino acids, which are used in vegetables, herbs and fermented foods in Korea, and in salads and vinegar materials in Europe.

Solutes that contribute to osmotic control in absolute halophytes are known as osmotic resistance organics. Typical osmotic agents that accumulate in cells include onium compounds such as amino acids proline and glycin betaine, mannitol, sorbitol, ononitol and monosaccharide trehalose .

It has been used for a long time as a folk remedy for visual loss, digestion failure, gastrointestinal illness, hepatitis, kidney disease, antidiabetic, anti-cancer, anti-hypertension, anti-hyperlipemia and skin whitening activity. There is not much research on this.

Korean Patent No. 0468073 discloses a composition comprising a green tea extract having anti-diabetic activity or isorhamnetin-3-O -? - D-glucoside isolated therefrom, Korean Patent No. 1212612 discloses a composition comprising (Isoquercitrin 6 " -O-methyloxalate) isolated from the cell line has antioxidant activity and an activity of inhibiting peroxidation.

As a result of intensive efforts to solve the above problems, the inventors of the present invention have found that extracts of Toyochimonoides and isorhamnetin isolated therefrom have the ability to prevent and treat nonalcoholic fatty liver disease and to complete the present invention .

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of nonalcoholic fatty liver disease originating from a natural substance,

Another object of the present invention is to provide a food composition for prevention or improvement of non-alcoholic fatty liver disease originating from Tungsten mushroom which is a raw material of food.

Another object of the present invention is to provide a method for isolating isorhamnetin from non-alcoholic bean curd refuse having the ability to prevent or treat non-alcoholic fatty liver disease.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating nonalcoholic liver disease, which comprises isorhamnetin derived from Salicornia spp . As an active ingredient.

In the present invention, the non-alcoholic fatty liver is selected from the group consisting of non-alcoholic liver inflammation, non-alcoholic liver fibrosis and non-alcoholic liver cirrhosis.

The present invention provides a food composition for preventing or ameliorating nonalcoholic fatty liver disease comprising isorhamnetin derived from Salicornia spp . As an active ingredient.

The present invention also relates to a process for the preparation of a composition comprising: (a) obtaining an extract of Trichoderma nodule; (b) fractionating the extract of Tungsten mulberry with a solvent; (c) fractionating the fractions of Chrysanthemum mildew solvent by chromatography to obtain a chromatographic fraction having inhibitory effect on hepatocyte inflammatory reaction and hepatocyte protective activity; And (d) isolating isorhamnetin using high performance liquid chromatography (HPLC) on said chromatographic fraction. The method of isolating isorhamnetin from Salicornia SPP . .

In the present invention, it is preferable that the extract of Tungtung nodule is fractionated with hexane, chloroform, ethyl acetate, butanol and water in order.

In the present invention, in the step of obtaining the chromatographic fraction, the solvent fraction is loaded on an adsorptive Diaion HP-20 column, and then eluted with acetone to obtain a first fraction having an inhibitory effect on the inflammatory reaction in the hepatocyte and hepatocyte protective activity , And loaded on a silica gel column. Then, methanol was sequentially added to the mobile phase of chloroform to elute the second fraction, which had an inhibitory effect on the inflammatory reaction in the hepatocyte and hepatocyte protective activity. The second fraction was loaded on an LH-20 column, And obtaining a third fraction having an inhibitory effect on the inflammatory response in the hepatocyte and a hepatocyte protective activity. The high performance liquid chromatography (HPLC) is characterized in that a mixture of methanol and water is used as a mobile phase.

Isorhamnetin derived from Salicornia spp . According to the present invention not only inhibits iNOS and COX-2 protein expression, which are typical inflammation markers in hepatocytes, in a concentration-dependent manner, but also exhibits typical cellular protection against oxidative stress By increasing the glutathione synthetase (GR, GCL) through Nrf2 activation, which is a transcription factor, it can be used for the prevention and treatment of nonalcoholic fatty liver diseases such as liver inflammation, liver fibrosis and cirrhosis.

(SEW-EA-B2), B: fraction (SEW-EA-B2-F2), C: fraction (SEW-EA-B2) Compound (1) purified in fraction (SEW-L-17).
Figure 2 is a isopropyl person netin compounds ESI-MS, ¹ H-NMR and ¹³ C-NMR spectrum is (A: iso person netin formula, B: ESI-MS, C : 1 H-NMR, D: 13 C-NMR ).
FIG. 3 shows the results of induction of C57BL / 6J mouse obesity via high fat diet.
FIG. 4 shows the results of a comparison between the weight of mouse liver extracted from the high fat and purified diet group and the high fat and tung fungus extract group, and the serum ALT level, which is an index of liver damage (A: mouse liver, B: mouse liver weight , C: mouse blood ALT).
FIG. 5 is a comparative measurement of the level of inflammation and fibrosis, which is a non-alcoholic fatty liver disease index in liver tissues in the high fat and purified diet group and the high fat and non-fat extract group (A: mouse hepatic tissue H & E staining and Masson's trichrome staining B: Steatosis, Inflammation, Ballooning results, C: NAFLD (non-alcoholic fatty liver disease) scoring score).
FIG. 6 shows the results of measurement of mRNA expression of inflammatory and hepatic fibrosis markers ( Il1b, Cxcl2, Ccl2, Cd68, Col1a1 ) in the liver tissues of the high fat and refined salt diet group and the high fat and noodle group extract diet groups.
FIG. 7 shows the results of confirming the inhibitory effect of nitric oxide (NO) release (A) and the inhibitory effect on the expression of iNOS protein (B) induced by human hepatocyte HepG2 cells stimulated with LPS of isolated isorhamnetin.
FIG. 8 shows the inhibition of COX-2 protein expression (A) and the inhibitory effect of NF-kB protein expression in the nucleus (B) induced by human hepatocyte-host HepG2 cells stimulated with LPS of isolated isorhamnetin to be.
FIG. 9 is a fluorescence microscopic photograph (B) showing isolametin isolated from the extract of Tongtung nodule inhibiting the production of ROS and the apoptosis of HepG2 cells treated with t-BHP (A) and observing the intracellular ROS elimination .
FIG. 10 shows the results of confirming the content (A) of glutathione (GSH) and the expression ability (B) of glutathione synthetase (GR, GCL) in hepatocytes treated with isorhamnetin isolated from the extract of Tungtung nodule.

In the present invention, we have sought to isolate active substances having excellent inhibitory activity against hepatocyte inflammatory response and hepatocyte-secreting activity,

In the present invention, it was confirmed that the extract of Toyochu-municipal noodles and isorhamnetin isolated therefrom have an inhibitory effect on hepatocyte inflammatory response and a hepatocyte-protective ability.

That is, in one embodiment of the present invention, it was confirmed that the extract of Tungtung-madi extract effectively inhibited liver damage, hepatitis and hepatic fibrosis caused by non-alcoholic fatty liver induced by high-fat diet and purified diet, Isolametin isolated by solvent fraction, chromatographic fractionation and high performance liquid chromatography (HPLC) not only inhibited protein expression of iNOS and COX-2, which are representative inflammation markers of hepatocyte inflammation, but also oxidative stress (GR, GCL) through the activation of Nrf2, a typical cytoprotective transcription factor, which is a counterproductive agent.

Accordingly, the present invention relates to a pharmaceutical composition for preventing or treating nonalcoholic fatty liver disease comprising isorhamnetin derived from Salicornia spp . As an active ingredient.

The pharmaceutical composition for preventing or treating nonalcoholic fatty liver disease of the present invention may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to isorhamnetin as an active ingredient, Release agents, sweeteners, binders, coating agents, swelling agents, lubricants, lubricants, and flavoring agents.

In addition to isorhamnetin, which is an active ingredient for administration, the pharmaceutical composition may further comprise at least one pharmaceutically acceptable carrier and may be formulated into a pharmaceutical composition. Formulations may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops, injectable solutions, and the like. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Further, if necessary, suitable binders, lubricants, disintegrants, coloring agents and the like may be included. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, Sodium benzoate, sodium acetate, sodium chloride and the like can be used. Examples of the disintegrating agent include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, and the like. Sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and the like may be used as the pharmaceutical carrier which is acceptable for formulation into a liquid solution And if necessary, usual additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added.

In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

The pharmaceutical composition of the present invention contains isorhamnetin which is isolated from a wild plant, Tongtung nodule, and has little toxicity and side effects. Therefore, for the purpose of preventing or treating non-alcoholic fatty liver disease and improving liver function, Can be used with confidence.

Also Salicornia (Salicornia according to the invention SPP.) Nonalcoholic fatty liver disease, for the prevention or treatment a pharmaceutical composition researcher, veterinarian, medical doctor or other clinical the jojikgye thought by animal or a biological or medical response in a human containing isopropyl person netin (isorhamnetin) of origin, as an active ingredient Or an amount of the pharmaceutical composition, i. E., A therapeutically effective amount, which is an amount that induces the reduction of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and the number of administrations of a composition containing isorhamnetin of the present invention as an active ingredient will vary depending on the desired effect. Thus, the optimal dosage to be administered can be readily determined by those skilled in the art and will vary with the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, The age, body weight, sex, diet, time of administration, route of administration and fraction of the composition, duration of treatment, concurrent medication, and the like.

In another aspect, the present invention relates to a food composition for preventing or ameliorating a nonalcoholic fatty liver disease comprising isorhamnetin derived from Salicornia spp . As an active ingredient.

In addition to containing isorhamnetin as an active ingredient, the food composition may further include various flavors, natural carbohydrates and the like used in ordinary food compositions.

Examples of the natural carbohydrate include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin. Conventional sugars and sugar alcohols such as xylitol, sorbitol and erythritol can be used. Examples of the flavoring agent include natural flavoring agents (e.g., tauromine), stevia extracts (e.g., rebaudioside A and glycyrrhizin), synthetic flavors (e.g., saccharin, aspartame, ) Can be used.

The food composition of the present invention may be formulated in the same manner as the above-described pharmaceutical composition and used as a functional food or may be added to various foods in the form of an additive. Foods to which the composition of the present invention can be added include beverages, meat, chocolates, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplement foods.

In addition to the isorhamnetin, which is an effective ingredient, the food composition can also contain flavorings such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates such as cheese and chocolate, An acid and its salt, alginic acid and its salt, an organic acid, a protective colloid thickener, a pH adjusting agent, a stabilizer, a preservative, a glycerin, an alcohol, and a carbonating agent used in a carbonated drink. In addition, the food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.

The health functional food in the form of the above food composition refers to the food prepared and processed using raw materials or ingredients having useful functions in accordance with Law No. 6727 on Health Functional Foods, It is meant to be ingested for the purpose of obtaining a beneficial effect for health use such as controlling nutrients or physiological action.

The above health functional foods may contain conventional food additives and, unless otherwise specified, conform to the general requirements of the Food Additives Ordinance approved by the Korea Food and Drug Administration, Judge by standards.

The additives added to the above-mentioned 'food additives' include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as persimmon extract, licorice extract, crystalline cellulose, high color pigment and guar gum; Mixed preparations such as a sodium L-glutamate preparation, a noodle-added alkaline agent, a preservative agent, and a tar coloring agent; .

The health functional food may be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and circles. The health functional food in the form of tablets may be prepared by granulating a mixture of isorhamnetin (isorhamnetin), an active ingredient of the present invention, with an excipient, a binder, a disintegrant and other additives by a conventional method, Alternatively, the mixture can be directly compression molded. In addition, a mating agent or the like may be added to the above-described healthful functional food in the form of tablet. The hard capsule of the capsule-type health functional food can be prepared by filling a mixture of isomerhenin, an active ingredient of the present invention, with an additive such as an excipient into a normal hard capsule, and the soft capsule is isarramine (isorhamnetin) with excipients such as excipients and the like, into a capsule such as gelatin. A plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, and the like may be added to the soft capsule. The ring-shaped health functional food can be prepared by molding a mixture of isorhamnetin, an active ingredient of the present invention, an excipient, a binder, a disintegrant, and the like by a conventionally known method, It can be applied to other skin preparations, or the surface can be coated with materials such as starch, talc. The granular health functional food may be prepared by granulating a mixture of isorhamnetin, an active ingredient of the present invention with an excipient, a binder, a disintegrant, etc., in a conventional manner, , A mating agent, and the like may be added.

In the present invention, nonalcoholic fatty liver may be exemplified by nonalcoholic liver inflammation, nonalcoholic liver fibrosis, nonalcoholic liver cirrhosis, and isorhamnetin derived from Salicornia spp . Inhibits nonalcoholic fatty liver disease disease In addition, there is strong antioxidant activity such as free radical and active oxygen scavenging, inhibition of lipid peroxidation, and cell protection against oxidative stress, and thus it can be widely used as a raw material for foods, cosmetics, pharmaceuticals, animal drugs and functional feeds.

In another aspect, the present invention relates to a method for preparing a composition comprising: (a) (b) fractionating the extract of Tungsten mulberry with a solvent; (c) fractionating the fractions of Chrysanthemum mildew solvent by chromatography to obtain a chromatographic fraction having inhibitory effect on hepatocyte inflammatory reaction and hepatocyte protective activity; And (d) isolating isorhamnetin using high performance liquid chromatography (HPLC) on said chromatographic fraction. The method of isolating isorhamnetin from Salicornia SPP . .

The extracts of Tungtung nodule are (a) washed and finely divided and then put into salt-free water and subjected to hot water extraction; (b) hot water extraction filtration; (c) The filtration residue was re-extracted at 100 ° C for 2 to 3 hours after putting it in an aqueous salt solution; (d) concentrating the re-extracted liquid and the primary hot water extract; (e) purifying the concentrate with 1 to 5% of activated carbon relative to the solid content; (f) drying (freezing or spraying). The non-saline water may be distilled water or tap water, and the non-saline water may be used in an amount of 1 to 5 times, preferably 1 to 3 times the volume of the tap water, and the hot water extraction is performed at about 100 ° C for 4 to 6 hours desirable.

In the present invention, the extract of Tungtung nodule is firstly fractionated with hexane, chloroform, ethyl acetate, butanol and water, Hexane fraction, chloroform fraction, ethylacetate fraction, butanol fraction and water fraction were obtained.

The step of obtaining the chromatographic fraction comprises performing Diaion HP-20 column chromatography, silica gel column chromatography and LH-20 column chromatography.

The elution solvent for the Diaion HP-20 column chromatography was 70% acetone. The elution solvent for the silica gel column chromatography was methanol, which was sequentially added to the chloroform mobile phase. The elution solvent for the LH-20 column chromatography was methanol .

The high-performance liquid chromatography (HPLC) can be carried out using a mixture of methanol and water as a mobile phase. For example, gradient conditions using methanol-grade methanol and tertiary distilled water, more specifically, methanol: : 55 to 100: 0 sequentially, and at a flow rate of 1 to 2 ml / min.

[Example]

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example 1: Isolation of the active ingredient compound 1 from the extract of Tungtung nodule

1-1. Preparation of hot water extract

And 10 kg of the crushed nodule outpost was subjected to hot water extraction at 100 ± 5 ° C for 5 hours by adding 20 L of non-salted water to an ultra-high vacuum concentrated water extractor (COSMOS 660, Kyung-Seo E & P, Korea). Next, the extract was filtered, and then 10 liters of water was added to the residue. The extract was re-extracted for 2 hours under the same conditions, and then mixed with the extract. The concentrate was then concentrated at 80 to 90 ° C to a salt content of 20% and a solid content of 40%. Finally, the concentrate was purified with 1 ~ 5% of activated carbon based on solid content and then lyophilized to prepare a hot water extract.

1-2. Acquisition of solvent fractions

374 g of the extract of Tungtung Nodi obtained in Example 1-1 was suspended in 1 L of water, 3 L of ethanol was added and the mixture was allowed to stand overnight at 4 DEG C. The resulting polymer precipitate was centrifuged (4 DEG C, 10000 rpm, 30 minutes) , And the filtrate was dried under reduced pressure to remove completely the ethanol. The solution was redissolved in 2 L of distilled water.

Next, 2 L of n-hexane was mixed with a 5-L separatory funnel, and the mixture was divided into two fractions, hexane layer and water layer. 2 L of chloroform was added to the water layer, and the mixture was divided into two portions with chloroform layer and water layer. Ethyl acetate was further added to the water layer, and the mixture was divided into two portions with ethyl acetate layer and water layer. Finally, 2 L of n-butanol was added to the aqueous layer, And water layer.

Next, each of the fraction layers was dried under reduced pressure to remove each organic solvent and lyophilized to obtain 0.8 g of hexane fraction, 0.9 g of chloroform fraction, 3.4 g of ethyl acetate fraction, 9.8 g of butanol fraction, The fractions (16.1 g) were prepared.

1-3. Column chromatography purification

The ethyl acetate fraction (SEW-EA) (3.4 g) having excellent antioxidative activity and hepatocyte protective activity among the organic solvent fractions prepared in Example 1-2 was dissolved in 50 ml of alkaline water (pH 10) to obtain an adsorbable diaion HP-20 The resin was adsorbed on the upper part of the first column (3.5 × 40 cm) filled with water, and then sequentially eluted with distilled water, 70% methanol and 70% acetone to obtain three fractions. 1.5 g of an acetone elution fraction (SEW-EA-B2) having excellent antioxidative activity and hepatocyte protective activity was dissolved in a chloroform solvent in which methanol was increased to a concentration gradient in a second column (2.8 x 40 cm) filled with polar silica gel, : Methanol was sequentially converted from 100: 0 to 70:30, and eluted at a flow rate of 0.3 ml / min to obtain five fractions of 350 ml each. The fraction (SEW-EA-B2-F2, 432 mg) having excellent antioxidative activity and hepatocyte protective activity was dissolved in 2 ml of methanol after drying under reduced pressure, and gel filtration with low molecular weight Sephadex LH-20 20 fractions (SEW-L-1 to SEW-L-20) were obtained by fractionation in 30 ml portions while flowing 95% methanol (flow rate 0.2 ml / min) . Finally, SEW-L-17 fraction having the highest antioxidative activity and hepatocyte protective activity among the 20 fractions was concentrated under reduced pressure and lyophilized to obtain 68 mg.

1-4. High Performance Liquid Chromatography (HPLC) Purification

68 mg of the SEW-L-17 fraction obtained in Example 1-3 was dissolved in 2 ml of methanol for HPLC, filtered through a 0.22 μm filter, and then subjected to high performance liquid chromatography to obtain a single substance having high antioxidative and hepatocyte protective activity Respectively. Analytical HPLC was performed using a model equipped with a Solbox Eclipse C18 analytical column (Zorbax Eclips, 5 μm, 4.5 × 250 mm, Agilent) (1260 Infinity, Agilent, USA) (Preparative HPLC (LC-forte / R, YMC, Japan) equipped with a double column (Triart C18, 20 mm × 150 mm, 5 μm, YMC, Japan) was used. Flow rate of 1 ~ 3ml / min was performed under gradient conditions using distilled water. Agilent, 1200 DAD detector or YMC-YUV-3400 UV detector was used and the two wavelength regions (260 and 330 nm) Compound 1 (32 mg) was obtained at a retention time of 13.5 minutes as a result of pure fractionation of the compounds using absorbance. Figure 1 shows SEW-EA-B2, SEW-EA-B2-F2, The analytical HPLC chromatogram of SEW-L-17 and the ultraviolet absorption wavelength spectrum of Compound 1 .

Example 2: Structural analysis of Compound 1 isolated from Tungtung nodule extract

The absorption maxima of Compound 1 isolated in Example 1-4 and adsorption shifts due to the addition of reagents were determined by dissolving each sample at a concentration of 1 mg / ml in methanol, and then measuring the concentration of each compound by using an ultraviolet spectrophotometer (Genesys 10S UV-VIS spectrophotometer , Thermo Scientific, USA). At this time, AlCl ₃ , NaOH, NaOAC, etc. were used to observe the changes of the ultraviolet absorption band due to the addition of the reagents.

Positive and negative scans were performed with an electronic foam ionization (ESI) mass spectrometer (LC-ESI mass spectrometer, AGILENT 1100, USA Micromass Quattro II) to determine the molecular weight of the compounds and high resolved MS was measured.

Compound 1 (10 mg) was completely dried, dissolved in DMSO- d ₆ (0.5 ml), injected into a 5 mm NMR tube, and analyzed with a Zeol model (JNM-ECA 600, Jeol, Japan) Respectively, and ¹ H-NMR was measured at 600 MHz and ¹³ C-NMR was measured at 150 MHz.

As a result of the measurement as described above, Compound 1 was identified as isorhamnetin, and its physicochemical properties were as follows.

(1) Molecular formula: C ₁₆ H ₁₂ O ₇

(2) Molecular weight: 316, ESI-MS: m / z 315.2 [M-H], m / z 631.2 [M +

(2) Melting point: 392 DEG C

(3) Appearance: yellow powder

(4) Solubility: soluble in methanol, ethanol, ethyl acetate, chloroform and insoluble in water

(5) TLC color development: FeCl ₃ (positive), bromocresol green (negative), UV light (positive, yellow green), anilindiphenylamine (negative), antimony (negative)

(6) UV absorption maximum wavelength region (MeOH, nm): 253, 267sh, 306sh, and 370, (+ NaOH): 273, 287sh, 326sh, 390, (+ AlCl 3): 253, 267sh, 306sh, and 423 _{(+ AlCl 3 + HCl):} 253, 267sh, 306sh, and 370; (+ NaOAc + H ₃ BO ₃ ) 254, 306, 382

(7) ¹ H and ^{13 C-NMR: 1 H NMR} (600 MHz, DMSO- d 6) δ ppm: 2.49 (1H, d, 3H), 3.83 (3H, s, OCH 3), 6.18 (1H (1H, d, 6H), 6.74 (1H, d, 8H) '-H), 9.43 (1H, s, OH), 9.75 (1H, s, OH), 10.76 (1H, s, OH), 12.46 (1H, s, 5-OH) ( Fig. 2C), ¹³ C- NMR (150 MHz, DMSO- d ₆ )? Ppm: 148.81 (2-C), 135.86 (3-C), 175.90 (4-C), 156.17 , 93.60 (8-C), 160.94 (9-C), 103.05 (10-C), 122.00 (1'-C), 111.65 (4'-C), 115.55 (5'-C), 121.72 (6'-C), 55.73 (OCH3)

(8) Chemical formula

Example 3: Determination of inhibitory effect of non-alcoholic fatty liver induced by high-fat diet and refined salt diet

3-1. High-fat diet of C57BL / 6J mice induced obesity by feeding

36 C57BL / 6J male 7-week-old mice were randomly divided into two groups as follows to induce obesity and insulin resistance by feeding each feed for 24 weeks. (HFD, D12492 Rodent Diet with 60 kcal% fat; Research Diets, n = 31) were divided into two groups: LFD, D12450B, Rodent Diet with 10 kcal% ( P <0.05) in the HFD-fed group (52.4 ± 0.5g) than in the LFD-fed group (40.5 ± 1.8g) when the LFD and HFD were consumed and the mean body weight was compared ).

The results showed that obese rats were well induced by high fat diet method. Therefore, non - alcohol fatty liver induction experiment was conducted in high fat diet induced rats by high fat diet and refined salt diet.

3-2. Evaluation of hepatic injury-improving efficacy induced by high-fat diet and refined salt ingestion

The high-fat diet was D12492 (Rodent Diet with 60 kcal% fat) purchased from Research Diets, USA. The salt concentration was calculated based on the adult daily intake recommended by the WHO (2 g sodium or 5 g salt) according to the previous literature, and the salt content was calculated as about 1% (NaCl content: about 56.41%) was about 1.77% of that of the high-fat diet, and that of one-week salt (refined salt, NaCl content : 99.52%) was about 1.00% of the high - fat diet, and the experiment was conducted based on this.

In addition, according to the previous literature that when the high fat diet was fed for a total of 36 weeks, fatty liver inflammation was evidently induced. The high fat diet was administered for 24 weeks, followed by the high fat and salt diet for 13 weeks. . As a result of analyzing organ weights after the autopsy, liver weight was significantly higher in the group fed high fat diet and purified salt (HFD + purified salt PS) than in the high fat diet group, In liver (HFD + Salicornia salt, SS) group, liver weight was significantly lower (P <0.05) than that of high fat diet and refined salt intake group. (Figs. 4A and 4B).

In addition, analysis of the plasma obtained after autopsy showed that alanine aminotransferase (ALT), an enzyme released into the blood when hepatocytes were damaged, was found to be higher in the high fat diet group And ALT levels were significantly lower in the high fat diet group than in the high fat diet group ( P <0.05) ( p <0.05) 4C).

3-3. Histological evaluation of non-alcoholic fatty liver disease (NAFLD)

In order to histologically confirm the non-alcoholic fatty liver disease improvement effect of the extract of Tungtung nodule, H & E staining and masson's trichrome staining were performed on liver tissue obtained after autopsy, and the fat test was analyzed.

(HFD + Salicornia salt, SS) supplemented with high-fat diet and high-fat diet (HFD + purified-salt PS) Showed efficacy. In the steatosis scoring category based on H & E staining, HFD + Salicornia salt (SS), a high fat diet group and HFD + purified salt PS group, ( P <0.094), and inflammation of the liver was also significantly inhibited.

In addition, when combined with Steatosis, Inflammation, and Ballooning, scoring of NAFLD (nonalcoholic fatty liver disease) improving efficacy shows that the extract of Tungtung nodule inhibits nonalcoholic lipidemia triggered by high fat diet and refined salt diet I could confirm.

For reference, the scoring criteria are as follows.

- Liver fibrosis progression score: 0 = Liver with fibrosis less than 10%, 1 = Liver with 10 to 33% fibrosis, 2 = 33 to 66% Liver with fibrosis, 3 =

- Score of progression of inflammation in the lobular lobe: 0 = 200 times when there is no lesion observed under field microscope, 1 = 200 times under field microscope, 2 = 200 times under field microscope. 4 to 4, 3 = 200 times Under field microscope, there are more than 4 lesions observed

3-4. Evaluation of the effects of extracts of Tungtung nodule on inflammation and fibrosis marker mRNA expression in liver tissue

MRNA expression of inflammatory and hepatic fibrosis markers (Il1b, Cxcl2, Ccl2, Cd68, Col1a1) in tissues was measured as follows. RNA was extracted from liver tissues obtained after autopsy using an RNA isolation kit (Ambion, RNA isolation kit, Life Technologies, Gaithersburg, MD, USA), and the RNA was extracted with NanoDrop ND-2000 (Thermo Fisher Scientific, Waltham, MA, USA) And cDNA was synthesized using 1 ^st strand c-DNA synthesis kit (Takara Bio Inc., Otsu, Japan). mRNA expression was confirmed by qRT-PCR (quantitative real-time polymerase chain reaction) using SYBR Green Master Mix (BioRad, Hercules, CA, USA) primers and 2 μl of synthesized c-DNA.

The primers before denaturation were denatured at 95 ° C for 3 minutes, and reacted for 44 cycles at 95 ° C for 10 minutes, 60 ° C for 30 minutes, and 72 ° C for 30 minutes. The mRNA content in the PCR reaction was measured using the CFX Connect ™ Real-Time PCR Detection System (BioRad, Hercules, Calif., USA). The primers used are as follows.

SEQ ID NO: 1: Ccl2 forward (5'-CCA CTC ACC TGC TGC TAC TCA T-3 ')

SEQ ID NO: 2: Ccl2 reverse (5'-TGG TGA TCC TCT TGT AGC TCT CC-3 ')

SEQ ID NO: 3: Il1b forward (5'-GGA GAA CCA AGC AAC GAC AAA ATA-3 ')

SEQ ID NO: 4: Il1b reverse (5'-TGG GGA ACT CTG CAG ACT CAA AC-3 ')

SEQ ID NO: 5: Cd68 forward (5'-ACC TGC TCA ACA TCA TGA AGG-3 ')

SEQ ID NO: 6: Cd68 reverse (5'-AGA TGG AGC TAT GCA GGT GG-3 ')

SEQ ID NO: 7: Cxcl2 forward (5'-CCA AGG GTT GAC TTC AAG AAC-3 ')

SEQ ID NO: 8: Cxcl2 reverse (5'-AGC GAG GCA CAT CAG GTA CG-3 ')

SEQ ID NO: 9: Col1a1 forward (5'-GCT CCT CTT AGG GGC CAC T-3 ')

SEQ ID NO: 10: Col1a1 reverse (5'-CCA CGT CTC ACC ATT GGG G-3 ')

(HFD + salicornia salt, SS), which consumed high-fat diet extract of Wanchungfumi extract (HFD + purified salt PS), showed higher levels of inflammation related markers ( cxcl2 , il1b ) and the expression of the fibrosis-related marker ( col1a1 ).

Example 4: Efficacy of isorhamnetin

4-1. Antioxidant activity investigation

In order to confirm the antioxidative activity of isorhamnetin identified as an active ingredient from the extract of Tungtung nodule in Example 2, scavenging activity, active oxygen scavenging ability and lipid peroxidation inhibiting activity of DPPH free radical were evaluated. At this time, alpha-tocopherol (vitamin E) and N-acetylcystein (NAC), a precursor of hepatocyte glutathione, which are antioxidant vitamins used for prevention and treatment of nonalcoholic fatty liver disease were used as a control group.

4-1-1: Measurement of glass radical scavenging ability

The antioxidant activity was determined according to the method of Bullois (Chen et al., 1999. J. Agric. Food Chem. 47. 2226-2228), 1,1-diphenyl-2-picrylhydrazide (1,1- diphenyl-2-picryl hydrazyl, DPPH, Sigma Co., USA). DPPH solution was prepared by dissolving 5 mg of DPPH in 50 ml of ethanol, adding 180 μl to 96-well microplate, adding 0-100 μg / ml of the sample, mixing for 5 seconds, And the absorbance reduction for the control group to which no sample was added at 517 nm was expressed as the free radical scavenging activity (%). The concentration of the substance required to eliminate 50% of the free radicals can be represented by the IC ₅₀ value, which means that the lower the value, the stronger the antioxidant capacity.

compound DPPH scavenging activity (IC ₅₀ value, μM) Isorhamnetine 10.6 Alpha-tocopherol 42.4 N-acetylcysteine 19.8

As shown in Table 1, it was confirmed that isorhamnetine isolated from Tungtung Mardi had about 4 times stronger than α-tocopherol and about 1.87 times stronger than N-acetylcysteine.

4-1-2: Measurement of reactive oxygen scavenging ability

The superoxide anion scavenging activity was measured by the oxidation of nitroblue tetrazolium (NBT) by active oxygen using an active oxygen generating system by xanthine / xanthine oxidase enzyme reaction. The concentration of the substance required to remove 50% of the active oxygen is measured using the absorbance (530 nm) change and is shown in Table 2 as the IC ₅₀ value.

compound The active oxygen scavenging activity (IC ₅₀ value, μM) Isorhamnetine 5.3 Alpha-tocopherol > 50 N-acetylcysteine 18.8

As shown in Table 2, it was confirmed that isorhamnetin isolated from Tungsten maddy had about 9 times stronger than alpha-tocopherol and about 3.5 times stronger than that of N-acetylcysteine.

4-1-3: Measurement of lipid peroxidation inhibition ability

The lipid peroxidation inhibition assay was performed by inducing lipid peroxidation by the Fe ²⁺ ascorbate system using microsomes obtained by ultracentrifugation (77000 g, 60 min, Hitachi RP 30) of the extracted rat hepatocytes as a lipid source, Aldehyde (MDA) was reacted with thiobarbituric acid (TBA) to quantitatively reduce the amount of MDA reduced by the sample. 200 μl of hepatic microsomal solution containing a sample in a test tube and 225 μl of sodium dodecyl sulfate (SDS) solution were added and mixed for 5 seconds with shaking. Then, 20% acetic acid, 75 μl of distilled water, 1.2% TBA 1 ml of the solution was added and the mixture was heated for 30 minutes. After cooling at room temperature for 30 minutes, the mixture was centrifuged at 3000 rpm for 20 minutes, and the supernatant was measured for absorbance at 532 nm to determine the lipid peroxidation inhibitory activity (%) of the control group to which no sample was added. Was expressed by IC ₅₀ value.

compound Lipid peroxidation inhibitory activity (IC ₅₀ value, μM) Isorhamnetine 20.5 Alpha-tocopherol 45.1 N-acetylcysteine 32.7

As shown in Table 3 above, it was confirmed that isorhamnetine isolated from Tungtung Mardi had a lipid peroxidation potency about 2.2 times higher than that of alpha-tocopherol and about 1.65 times higher than N-acetylcysteine.

4-2. By LPS  Stimulated Human HepG2  Concentration-dependent NO emission of isopramenine active ingredient isolated from Tungsten nodules in cells Inhibition  And iNOS  Protein expression Low performance  Measure

In order to analyze the anti-inflammatory effect of isorhamnetin isolated from Tungtung maddy, the nitrite (NO) produced in human HepG2 cells stimulated with LPS (100 ng / ml), a hepatocyte inflammation inducer, . Nitrite (NO) was measured by NO assay using Griess reagent.

As a result, as shown in FIG. 7A, the nitrite (NO) induced by LPS in HepG2 cells was increased by about 7.5-fold compared with the control group of 3.35 ± 0.52 μM at 25.13 ± 0.75 μM, The concentration of nitrite (NO) was decreased by 20.59 ± 0.68μM, 12.08 ± 0.46μM and 5.01 ± 0.09μM, respectively, depending on the concentrations (10, 25 and 50μM)

Immunoblotting experiments were carried out to confirm that isoramicin inhibits protein expression of iNOS, a nitrate (NO) synthesis inducer, in a concentration-dependent manner. In order to analyze the expression pattern of iNOS protein, isolametin and LPS were simultaneously treated with HepG2 cells and cultured for 18 hours. Then, cells were lysed, and proteins were quantitated (Bradford assay, BioRad Laborotories, Hercules, CA). Next, the same amount (20 μg) of the protein was loaded on a 12% SDS-PAGE gel (Min Bio-Rad, Hets, UK) and subjected to electrophoresis. The separated proteins were transferred to a nitrocellulose membrane (Hybond-C Extra, Amersham), bound to a primary antibody for iNOS (Santa Cruz Biotechnology, Santa Cruz, Calif.) And then incubated with a secondary antibody (HRP-conjugated goat anti-rabbit immunoglobulin G antibody, Sigma). iNOS protein expression was confirmed by β-actin and chemiluminescence detection kit (Amersham Bioscience).

As shown in FIG. 7B, it was confirmed that the iNOS protein amplified intracellularly by LPS treatment was decreased in a concentration-dependent manner by isorhamnetin treatment. In particular, 50 μM isorhamnetin showed iNOS protein expression amplified by LPS, As shown in Fig.

4-3. Measurement of NF-kB and COX-2 Protein Expression of Isolametin Components Isolated from Tung Mun in Human HepG2 Cells

Expression of COX-2 and NF-kB overexpressed in human HepG2 cells stimulated with LPS (100 ng / ml), a hepatocyte inflammation inducer, in order to analyze the inhibitory effect of isorhamnetin on liver inflammation induced by nonalcoholic fatty liver Were inhibited by isorhamnetin. COX-2 (cyclooxygenase type 2), an enzyme involved in the production of inflammation, is an enzyme that produces Prostaglandin E ₂ , an intracellular inflammatory substance. COX-2 protein expression is used as a biomarker of a typical inflammatory factor.

In order to examine the effect of isorhamnetin on hepatocyte inflammation, HepG2 cells were treated with LPS (100 ng / ml) and LPS (100 ng / ml) to induce oxidative stress in the cells or stimulate the cells with inflammatory factors such as LPS. Isolametin was treated for each concentration and cultured for 18 hours. After culturing, the protein of each cell was quantified and Western blot was performed to confirm the expression pattern of COX-2 protein. The result is shown in FIG. 8A.

The COX-2 gene promoter region is known to have NF-kB activation due to oxidative stress or inflammatory stimuli, as there is a region where p65 binds as an activation-phosphorylated NFkB fragment. Therefore, NFkB activated by LPS treatment was confirmed by the expression of phospho-p65 protein in the nucleus. To confirm whether isorhamnetin also inhibited NFkB activation, HepG2 cells were treated with LPS (100 ng / ml) and isorhamnetin After incubation for 18 hours, the nuclei of each cell were separated and the expression of phospho-p65 in the nucleus was confirmed by Western blotting.

To separate the nuclei from HepG2 cells, a nuclear extraction kit (Active Motif, Carlsbad, Calif., USA) was used at 4 ° C. The separated nuclei were lysed and centrifuged at high speed (21,000 g, 30 min) After protein quantification, electrophoresis was carried out and the results are shown in FIG. 8B.

As shown in FIG. 8B, it was confirmed that isorhamnetin inhibits NF-kB activation in a concentration-dependent manner.

4-4. Concentration-dependent inhibition of ROS production and cell death by the active ingredient of isorhamnetin isolated from Tungtung nodule in human HepG2 cells treated with t-BHP

One of the representative mechanisms of nonalcoholic fatty liver disease is that active oxygen produced during fatty oxidation caused by accumulation of liver fat by high fat diet causes inflammation or direct toxicity due to oxidative stress causes cell necrosis It is known. Therefore, it is possible to directly detect ROS in HepG2 cells and to confirm whether or not isarramine is capable of eliminating and suppressing intracellular ROS produced by the treatment of t-BHP (tertiary butyl hydroperoxide), which is a strong peroxide, Respectively.

B cells treated with isorhamnetin at the concentration and 1 mM t-BHP were added to the cells not pretreated with isorhamnetine. After 18 hours, intracellular ROS content and cell necrosis were observed. The intracellular ROS content of the HepG2 cells treated with t-BHP on 6-well plates was determined by DCFH-DA (2 ', 7'-chlorofluorescein) , 7'-chlorofluorescein diacetate) was measured with a fluorescence meter (TECAN, San Jose, Calif.) At a wavelength between 485 and 530 nm.

Cell viability of cell necrosis was determined by MTT assay. That is, HepG2 cells were cultured in 6-wells, treated with isorhamnetin for 18 hours at a concentration, the medium containing isorhamnetin was removed, the cells were washed with PBS, and 1 mM t-BHP was added Respectively.

From FIG. 9A, the cell survival rate of HepG2 cells treated with t-BHP alone was significantly decreased to 20.2% ± 0.88 due to cell necrosis. However, cells of the medium supplemented with isorhamnetin by the pretreatment showed a The survival rate gradually increased, and the cell survival rate was nearly 100% at the 50 μM treatment of isorhamnetin, and the isoxanthin-induced inhibition of oxidative stress induced cell necrosis was confirmed.

In addition, ROS was rapidly increased in the cells treated with t-BHP alone, but the production of intracellular ROS was suppressed in a concentration-dependent manner in the cells treated with isorhamnetine, and at the level of 50 μM, It was confirmed that it was lowered. 9B is a fluorescence microscope photograph of the intracellular ROS.

4-5. Measurement of glutathione synthetase (GR, GCL) protein induction and intracellular GSH content enhancement by activation of Nrf2 transcription factor of isorhamnetin in human HepG2 cells

(GSH) and glutathione synthetases (GR, GCL) and the transcription of these enzyme proteins when isorhamnetin was treated with cells to study the mechanisms of hepatocyte inflammation mitigation and cytoprotective effects of isorhamnetin And Nrf2, respectively. The content of glutathione (GSH), known as a hepatocyte protective factor, is closely related to the amount of protein expressed by glutathione reductase (GR) and gamma glutamylcysteine ly ace (GCL), and recently, the precursor of glutathione and glutathione N-acetylsystein is used in the treatment of fatty liver.

HepG2 cells were treated with isorhamnetin at different concentrations (0, 2.5, 5, 10, 25, 50, 100 μM) for 18 hours and the intracellular glutathione (GSH) content was measured. The results are shown in FIG. 10A.

As shown in FIG. 10A, it was confirmed that isorhamnetine increases intracellular glutathione (GSH) content in a concentration-dependent manner, and that glutathione increases more than twice as much as that of the control group when treated with a concentration of 50 μM or more.

Therefore, we investigated whether the increase of glutathione in HepG2 cells by isorhamnetin is due to the Nrf2 transcription factor activity by isorhamnetin and the protein expression of glutathione synthetase, GCL and GR, by the phase II gene activation, the downstream genes of Nrf2 Western blotting was carried out after treatment of isorhamnetin with concentration (0, 2.5, 5, 10, 25, 50, 100 μM) for 18 hours and the results are shown in FIG. 10B. Cells treated with isorhamnetin and control cells were lysed for Westin blotting and proteins were quantified (Bradford assay, BioRad Laborotories, Hercules, CA). Next, the same amount (20 μg) of the protein was loaded on a 12% SDS-PAGE gel (Min Bio-Rad, Hets, UK) and subjected to electrophoresis. The separated proteins were transferred to a nitrocelluose membrane (Hybond-C Extra, Amersham), bound to a primary antibody against GR and GCL (Santa Cruz Biotechnology, Santa Cruz, Calif.), conjugated goat anti-rabbit immunoglobulin G antibody, Sigma). GCL and GR protein expression was confirmed by β-actin and chemiluminescence detection kit (Amersham Bioscience). It was confirmed that isorhamnetin increases GCL and GR expression, which are glutathione-synthesizing enzymes, in a concentration-dependent manner.

As shown in FIG. 10B, the activation of Nrf2 transcription factor by isorhamnetine was confirmed by the expression amount of Nrf2 protein present in the nucleus. It was found that isorhamnetin was activated by intranuclear transfer of Nrf2 transcription factor at a concentration of 10 μM or more, and Nrf2 activation induced by isorhamnetin was similar to GCL and GR expression.

Therefore, the increase of glutathione in hepG2 cells by isorhamnetin is caused by the synthesis of GCL and GR enzyme proteins by activation of Nrf2 transcription factor, which is effective for relieving inflammation and treatment of fatty liver cells by reducing oxidative stress caused by fatty acid oxidation in hepatocytes .

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Phyto Corporation <120> Pharmaceutical Composition for Prevention or Treatment of          Non-alcoholic Fatty Liver Disease Containing Isorhamnetin derived          from Salicornia europaea <130> P16117 <160> 10 <170> KoPatentin 3.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Ccl2 forward primer <400> 1 ccactcacct gctgctactc at 22 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Ccl2 reverse primer <400> 2 tggtgatcct cttgtagctc tcc 23 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Il1b forward primer <400> 3 ggagaaccaa gcaacgacaa aata 24 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Il1b reverse primer <400> 4 tggggaactc tgcagactca aac 23 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Cd68 forward primer <400> 5 acctgctcaa catcatgaag g 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cd68 reverse primer <400> 6 agatggagct atgcaggtgg 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Cxcl2 forward primer <400> 7 ccaagggttg acttcaagaa c 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cxcl2 reverse primer <400> 8 agcgaggcac atcaggtacg 20 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Col1a1 forward primer <400> 9 gctcctctta ggggccac 18 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Col1a1 reverse primer <400> 10 ccacgtctca ccattgggg 19

Claims (7)

delete delete delete (a) obtaining an extract of Tungsten mulberry;
(b) fractionating the extract of Tungsten mulberry with a solvent;
(c) fractionating the fractions of Chrysanthemum mildew solvent by chromatography to obtain a chromatographic fraction having inhibitory effect on hepatocyte inflammatory reaction and hepatocyte protective activity; And
(d) isolating isorhamnetin using high performance liquid chromatography (HPLC) on said chromatographic fraction. The method of isolating isorhamnetin from Salicornia spp .
5. The method according to claim 4, wherein the extract of Tungtung nodule is obtained by isolating isorhamnetin from Salicornia spp ., Characterized in that the extract of Tungtung nodule is sequentially fractionated with hexane, chloroform, ethyl acetate, butanol and water. Way.
5. The method of claim 4, wherein the step of obtaining the chromatographic fraction comprises: loading the solvent fraction into an adsorbable Diaion HP-20 column; then eluting the column with acetone to obtain a first fraction having inhibitory activity against inflammation in hepatocytes and hepatocyte- And then loaded on a silica gel column. Then, methanol was sequentially added to the mobile phase of chloroform to elute the second fraction, which was then loaded on an LH-20 column, To obtain a third fraction having an inhibitory effect on the inflammatory reaction in the hepatocyte and hepatocyte protective activity. The high-performance liquid chromatography (HPLC) is Salicornia (Salicornia SPP.) Method of separating iso-derived person netin (isorhamnetin), characterized in that is carried out with a mobile phase of methanol and water mixture.
The method of claim 4, wherein the high performance liquid chromatography (HPLC) is tungtung, characterized in that is carried out by a mobile phase of methanol and water mixture node (Salicornia SPP .) Isomerane (isorhamnetin).

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