KR20180000703A - Salicornia SPP. Extracts Containing Isorhamnetin Having Effect for Prevention or Treatment of Non-alcoholic Fatty Liver Disease and Sodium chloride - Google Patents

Abstract

The present invention relates to an extract of Salicornia SPP., and more specifically, to an extract of Salicornia SPP. comprising sodium chloride and isorhamnetin capable of preventing or treating non-alcoholic fatty liver diseases. The isorhamnetin derived from Salicornia SPP. according to the present invention decreases the expression of representative inflammatory markers in liver cells, iNOS and COX-2 proteins in a concentration-dependent manner, and also increases glutathione synthesis enzymes (GR, GCL) through the activation of a representative cell protective transcription factor that fights oxidative stress, Nrf2, and thus may be used in the prevention and treatment of non-alcoholic fatty liver diseases including hepatic inflammation, liver fibrosis, hepatocirrhosis, and the like.

Description

Salicornia SPP. Containing isoramnetin and sodium chloride, which are capable of preventing or treating non-alcoholic fatty liver disease {Salicornia SPP. Extracts Containing Isorhamnetin Having Effect for Prevention or Treatment of Non-alcoholic Fatty Liver Disease and Sodium chloride}

The invention Salicornia containing isopropyl person netin and sodium chloride in isopropyl ability person Salicornia containing netin and sodium chloride (Salicornia SPP.) Relates to the extract, more particularly non alcoholic fatty liver disease prevention or treatment (Salicornia SPP . ) Relates to the extract.

According to the statistics on the causes of death of the National Statistical Office in 2014, cancer, heart disease, and vascular disease, the three major causes of death of Koreans, account for 47.4% of all deaths, and it is reported that death is followed by pneumonia, diabetes, and liver disease. In particular, 6,635 people died from liver disease, accounting for 13.1% per 100,000 population. Fatty liver is caused by the accumulation of excess fat (mainly triglycerides) in the liver. In general, fatty liver is diagnosed if more than 5% of the weight of the liver is accumulated (medical information on the National Health Information Portal). Fatty liver is a very common disease, so there is a study that most (60-80%) of patients who visit the hospital for liver function abnormalities are fatty liver, and 20-30% of the total population is fatty liver.

In addition, it was found that 3 out of 10 domestic adults had fatty liver symptoms, and as a result of a survey of the proportion of patients in the gastroenterology department at Samsung Seoul Hospital in 2014, 35.8% of all age groups were in their 30s, 34.3% in their 40s, and 30.6 in their 50s. %, followed by 24.6% in their 60s, and 24.1% in their 20s, and the percentage of fatty liver in their 30s was the highest. As a result of analyzing the information of 750,000 adults who received medical examination from 1988 to 2014 by the Korean Liver Association, it was found that 70% of diabetic patients, 52% of hypertensive patients, and 38% of metabolic syndrome patients suffered from fatty liver. In addition to the liver-related treatment market, there is a growing interest in products for improving liver function and demand from consumers in the health functional food market.

Fatty liver can be divided into alcoholic fatty liver due to heavy drinking and non-alcoholic fatty liver disease (NAFLD), which is caused by obesity due to high fat diet regardless of alcohol, and 15% of nonalcoholic fatty liver is steatohepatitis. And hepatic fibrosis, 5% of steatohepatitis transitions to cirrhosis or liver cancer. In addition, it is known that non-alcoholic fatty liver (NAFLD) is induced by overdose of drugs and hyperlipidemia in which the amount of total cholesterol and triglycerides in blood vessels is increased above normal levels by lowering the liver detoxification function and lipolytic function.

Statin drugs such as gemfibrozil and atorvastatin, drugs prescribed for the prevention and treatment of non-alcoholic fatty liver disease, have been reported to damage mitochondria in muscles. It is not known to be suitable. One of the mechanisms of development of hepatitis and hepatic fibrosis, which are representative diseases of non-alcoholic fatty liver, is that free radicals caused by fatty acid oxidation in hepatocytes activate NFkB, a central transcription factor for inflammation-related signaling pathways, and related inflammatory genes, COX-2 and iNOS. Causes the synthesis of

Vitamin E (tocopherol) and vitamin C (ascorbic acid) are effective in preventing fatty liver and liver function levels and biopsy of patients with steatohepatitis to some extent, and are precursors of glutathione (GSH), an antioxidant that naturally exists in hepatocytes. It is known that various drugs (N-acetylcysteine, S-adenosyl-methionine [SAM]) can be used to treat fatty liver due to its antioxidant activity that increases glutathione in the liver. Intrahepatic glotathione (GSH) is synthesized from gamma-glutamylcysteine lyase (GCL), a central synthase, and is reduced from oxidized GSSG by the action of glutathione reductase (GR). . The increase of glutathione synthase in hepatocytes, such as glutathione reductase (GR) and gamma glutamylcysteine lyase (GCL), activates Nrf2, a representative transcription factor in the maintenance of cell homeostasis and cell protection mechanisms. It proceeds through. Therefore, it is suggested that antioxidant components derived from natural products that can inhibit the activation of NFkB, an inflammatory factor, and activate cytoprotective and protective Nrf2 transcription factors can also show efficacy in the prevention and treatment of steatohepatitis disease. Therefore, there is a need to develop a new drug derived from natural products for the prevention and treatment of non-alcoholic fatty liver, which has no side effects and is inexpensive and has excellent therapeutic effect.

Korean Patent Publication No. 2015-0103847 relates to a composition for the prevention or treatment of non-alcoholic fatty liver disease containing a brown flower extract as an active ingredient, and the brown flower extract is a non-alcoholic fatty liver such as fat accumulation and deformation of liver tissue due to lipid accumulation. It was disclosed that it can alleviate the pathological findings caused by and reduce blood lipid content, and inhibit the expression of PPARγ and C/EBPα, which are adipocyte transcriptional regulators, and SREBP-1, a transcription factor that regulates fatty acid biosynthesis. , Separation of the active ingredient was not attempted.

Korean Patent No.0867320 relates to a composition for preventing or treating liver fibrosis (liver cirrhosis) using water extracts of 13 kinds of herbal medicines. Although bilirubin, AST, and ALT were investigated and the content of MDA, which is a lipid peroxide substance in liver tissue, was analyzed, no studies were attempted on the active ingredient.

On the other hand, Salicornia europaea is a representative salt-growing plant that inhabits large amounts in the tidal flats of the west coast of Korea, and has strong salt resistance, so it can grow without loss of growth even in sand-cultivation with 200 mM NaCl added, and salts are absolutely necessary for growth. It is an absolute salt plant (Obligatory halophyte or True halophyte). In addition to sodium, Tungsuki contains a large amount of minerals such as magnesium, potassium, and iron, and abundant amino acids. In Korea, it has been used as vegetables, herbs, fermented foods, etc., and in Europe, it is used as ingredients for salads and vinegar.

Solutes that contribute to osmotic regulation in absolute salt plants are known as osmotic resistance organic substances. Representative osmotic resistance substances accumulated in cells include onium compounds such as amino acids proline and glycin betaine, sugar alcohols such as mannitol, sorbitol, ononitol, and monosaccharides trehalose. It is known as.

As a folk medicine for a long time, lumps have been used for vision loss, indigestion, gastrointestinal disease, hepatitis, kidney disease, etc., and only studies have been conducted on anti-diabetes, anti-cancer, anti-hypertension, anti-hyperlipidemia, and skin whitening activity. There was not much research on

Korean Patent No. 0468073 discloses a composition comprising a green tea extract having antidiabetic activity or isoramnetin-3-O-β-D-glucoside isolated therefrom, and Korean Patent No. 1212612 discloses It has been disclosed that isoquercitrin 6''-O-methyloxalate isolated from isoquercitrin 6''-O-methyloxalate has antioxidant activity and peroxide production inhibitory activity.

Accordingly, the present inventors have made diligent efforts to solve the above problems, and as a result of confirming that the lumpy dulcis extract and isorhamnetin isolated therefrom have the ability to prevent and treat non-alcoholic fatty liver disease, to complete the present invention. Became.

It is an object of the present invention to provide a natural extract having the ability to prevent non-alcoholic fatty liver disease and a food and feed composition comprising the same.

In order to achieve the above object, the present invention provides a Salicornia SPP. extract containing isorhamnetin and sodium chloride (NaCl) that are capable of preventing or treating non-alcoholic fatty liver disease.

In the present invention, the non-alcoholic fatty liver is characterized in that it is selected from the group consisting of non-alcoholic hepatitis, non-alcoholic liver fibrosis, and non-alcoholic liver cirrhosis.

The present invention also provides a food and feed composition comprising an extract of Salicornia SPP. containing isoramnetin and sodium chloride (NaCl), which are capable of preventing non-alcoholic fatty liver disease.

Isorhamnetin derived from Salicornia SPP according to the present invention not only concentration-dependently inhibits the expression of iNOS and COX-2 proteins, which are representative inflammatory markers in hepatocytes, but also protects representative cells against oxidative stress. By increasing glutathione synthase (GR, GCL) through activation of the transcription factor Nrf2, it can be used for the prevention and treatment of non-alcoholic fatty liver diseases such as hepatitis, liver fibrosis, and cirrhosis.

1 is an HPLC chromatogram of a fraction and compound 1 (isoramnetin) isolated from a serrata extract (A: fraction (SEW-EA-B2), B: fraction (SEW-EA-B2-F2), C: Compound 1 purified from the fraction (SEW-L-17).
2 is an ESI-MS, ¹ H-NMR and ¹³ C-NMR spectrum of an isoramnetin compound (A: isoramnetin chemical formula, B: ESI-MS, C: ¹ H-NMR, D: ¹³ C-NMR ).
3 is a result showing the induction of obesity in C57BL/6J mice through high fat diet.
Figure 4 is a result of comparing and measuring the weight of mouse livers extracted from the high fat and purified salt diet group and the high fat and sorghum extract diet group and blood ALT level, which is an indicator of liver damage (A: mouse liver properties picture, B: mouse liver weight , C: mouse blood ALT).
5 is a result of comparing and measuring the level of inflammation and fibrosis, which are indicators of non-alcoholic fatty liver disease, in liver tissues in the high fat and purified salt diet group and the high fat and lump extract diet group (A: mouse liver tissue H&E staining photograph and Masson's trichrome staining Photograph, B: Steatosis, Inflammation, Ballooning result, C: NAFLD (non-alcoholic fatty liver disease) scale score).
6 is a result of measuring the mRNA expression of inflammation and hepatic fibrosis markers (Ilb, Cxcl2, Ccl2, Cd68, Col1a1 ) in liver tissues of the high fat and purified salt diet group and the high fat and gallaria extract diet group.
7 is a result of confirming the ability to inhibit the amount of nitrite (NO) release (A) and the ability to inhibit iNOS protein expression (B) induced in HepG2 cells, which is a human hepatocyte cell line stimulated with LPS, of isoramnetin isolated from A.
Figure 8 is a result of confirming the ability to inhibit the expression of COX-2 protein induced in HepG2 cells, which is a human hepatocyte cell line stimulated with LPS, of isoramnetin isolated from the salicorniflora extract (A) and the ability to inhibit NF-kB protein expression in the nucleus (B) to be.
FIG. 9 is a fluorescence micrograph (B) in which isoramnetin isolated from the stalk extract inhibits the generation and apoptosis of ROS produced in HepG2 cells treated with t-BHP (A) and observes the elimination of intracellular ROS (B). .
FIG. 10 is a result of confirming the content (A) of glutathione (GSH) and the expression ability (B) of glutathione synthetase (GR, GCL) in isoramnetin-treated hepatocytes isolated from the P.

In the present invention, it was attempted to isolate an active substance having excellent ability to inhibit inflammatory reactions in hepatocytes and protect hepatocytes from a sore not widely studied so far.

In the present invention, it was confirmed that the extract and isoramnetin isolated therefrom have the ability to inhibit inflammatory reactions in hepatocytes and protect hepatocytes.

That is, in one embodiment of the present invention, it was confirmed that the hot-water extract of Salicornia chinensis effectively inhibits the process of liver damage, hepatitis, and liver fibrosis caused by non-alcoholic fatty liver triggered in a high-fat diet and a refined salt diet. Isoramnetin, isolated by solvent fractionation, chromatography fractionation, and high-performance liquid chromatography (HPLC), not only concentration-dependently inhibited the protein expression of iNOS and COX-2, which are representative inflammation markers in hepatocytes, but also against oxidative stress. It was confirmed that glutathione synthesizing enzymes (GR, GCL) were increased through the activation of Nrf2, a representative cytoprotective transcription factor that counteracts.

Accordingly, the present invention relates to an extract of Salicornia SPP. containing isorhamnetin and sodium chloride (NaCl) that are capable of preventing or treating non-alcoholic fatty liver disease.

The extract of Salicornia SPP. of the present invention contains isorhamnetin isolated from the salt plant, Salicornia SPP. There is little toxicity and side effects, thus preventing or treating non-alcoholic fatty liver disease and improving liver function It can be used with confidence even when taken for a long time for the purpose of.

The invention also relates to in another aspect, the food or feed composition comprising the Salicornia (Salicornia SPP.) Extract containing isopropyl person netin and sodium chloride (NaCl) with capability non alcoholic fatty liver disease prevention.

The food and feed composition may additionally contain various flavoring agents and natural carbohydrates used in conventional food compositions in addition to containing isorhamnetin as an active ingredient.

The natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.), polysaccharides (eg, dextrin, cyclodextrin), and the like. Conventional sugars or sugar alcohols such as xylitol, sorbitol, and erythritol may be used. As the flavoring agent, natural flavoring agents (eg, taumatin, etc.), stevia extracts (eg, rebaudioside A, glycyrrhizin, etc.), synthetic flavoring agents (eg, saccharin, aspartame, etc.) ) Can be used.

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

In addition, the food composition includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), in addition to the active ingredient isorhamnetin. Acids and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like may be further added. In addition, the food composition of the present invention may contain flesh for the production of natural fruit juice and fruit juice beverages and vegetable beverages.

Among the forms of the food composition, the term "health functional food" refers to a food manufactured and processed using raw materials or ingredients having useful functions for the human body pursuant to No.6727 of the Health Functional Food Act, and with respect to the structure and function of the human body. It refers to ingestion for the purpose of obtaining beneficial effects for health purposes such as controlling nutrients or physiological effects.

The above health functional food may contain ordinary food additives, and whether it is suitable as a food additive is determined according to the general rules and general test methods for food additives approved by the Food and Drug Administration, unless otherwise specified. It is judged according to the criteria.

Examples of additives listed in the'food additives' include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as reduced pigment, licorice extract, crystalline cellulose, high color pigment, and guar gum; Mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservatives preparations, and tar coloring preparations; Etc.

The health functional food can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and pills. For health functional food in tablet form, a mixture of isorhamnetin, an active ingredient of the present invention, mixed with an excipient, a binder, a disintegrant, and other additives, is granulated by a conventional method, and then a lubricant is added and compression molded. Alternatively, the mixture may be directly compression molded. In addition, a mating agent or the like may be added to the health functional food in the form of a tablet, if necessary. Among the capsule-type health functional foods, hard capsules can be prepared by filling a conventional hard capsule with a mixture of isorhamnetin, an active ingredient of the present invention, with an additive such as an excipient, and the soft capsule is isoramnetin. (Isorhamnetin) can be prepared by mixing a mixture of excipients and other additives into capsules such as gelatin. Plasticizers such as glycerin or sorbitol, colorants, preservatives, etc. may be added to the soft capsules as needed. The cyclic 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, etc., by conventionally known methods. It may be coated with another coating agent, or the surface may be coated with a material such as starch or talc. The health functional food in the form of granules can be prepared in granular form by a mixture of isorhamnetin, an active ingredient of the present invention, an excipient, a binder, a disintegrant, etc., by a conventionally known method. , A mating agent, etc. may be added.

In the present invention, non-alcoholic fatty liver may exemplify non-alcoholic hepatitis, non-alcoholic liver fibrosis, non-alcoholic liver cirrhosis, and the like, and isorhamnetin derived from Salicornia SPP. In addition, since it has strong antioxidant activity such as scavenging free radicals and active oxygen, inhibiting lipid peroxidation, and cell protection against oxidative stress, it can be widely used as a raw material for food, cosmetics, pharmaceuticals, animal medicines, and functional feed.

[Example]

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Example 1: Isolation of Compound 1 as an Active Ingredient from the Extract of Sprouts.

1-1. Preparation of hot water extract

After washing, 10 kg of chopped sprouts were extracted with hot water at 100±5° C. for 5 hours by adding 20ℓ of unsalted water with an ultra-high-speed vacuum condensed water extractor (COSMOS660, Gyeongseo E & P, Korea). Next, after the extract was filtered, 10 L of water was added to the residue, extracted again for 2 hours under the same conditions, mixed with the extract obtained first, and then concentrated to 20% salinity and 40% solid content at 80-90°C. Finally, the concentrate was purified with 1 to 5% activated carbon compared to the solid content, and then freeze-dried to prepare a hot water extract.

1-2. Solvent Fraction Acquisition

After suspending 374 g of the hot-water extract obtained in Example 1-1 in 1 liter of water, 3 liter of ethanol was added and allowed to stand overnight at 4°C. The resulting polymer precipitate was centrifuged (4° C., 10000 rpm, 30 minutes) and filtered under reduced pressure. Then, the filtrate was dried under reduced pressure to completely remove ethanol, and the solution was redissolved in 2 L of distilled water.

Next, 2 liter of n-hexane was mixed in a 5 liter volume separatory funnel, and the mixture was divided into a hexane layer and an aqueous layer twice. 2 L of chloroform was added to the aqueous layer, divided into a chloroform layer and an aqueous layer twice, ethyl acetate was added to the aqueous layer, and fractionated twice into an ethyl acetate layer and an aqueous layer, and finally, 2 L of n-butanol was added to the aqueous layer, followed by a butanol layer. It was partitioned twice into a fruit layer.

Next, each of the fractional layers was dried under reduced pressure to remove each organic solvent and lyophilized, followed by a hexane fraction (0.8g), a chloroform fraction (0.9g), an ethyl acetate fraction (3.4g), a butanol fraction (9.8g) and water. A fraction (16.1 g) was prepared.

1-3. Column chromatography refine

Among the organic solvent fractions prepared in Example 1-2, ethyl acetate fraction (SEW-EA) (3.4 g) having excellent antioxidant activity and hepatocyte protective activity was dissolved in 50 ml of alkaline water (pH 10) and adsorbable diaion HP-20 After adsorbing to the upper part of the first column (3.5 × 40 cm) filled with resin, three fractions were obtained by sequentially eluting with distilled water, 70% methanol and 70% acetone. Among them, 1.5 g of the acetone eluted fraction (SEW-EA-B2), which has excellent antioxidant activity and hepatocyte protective activity, is a chloroform solvent in which methanol is increased in a concentration gradient in the second column (2.8 × 40 cm) filled with polar silica gel, more specifically chloroform. : The methanol ratio was sequentially converted from 100:0 to 70:30, eluting at a flow rate of 0.3 ml/min to obtain 5 fractions of 350 ml each. And the fraction (SEW-EA-B2-F2, 432 mg) having excellent antioxidant activity and hepatocyte protective activity was dissolved in 2 ml of methanol after drying under reduced pressure, and gel filtration for low molecular weight Sephadex LH-20 was filled. Introduced into the third column (2 × 33 cm) and fractionated by 30 ml while flowing 95% methanol (flow rate 0.2 ml/min), and a total of 20 fractions (SEW-L-1 ~ SEW-L-20) were collected. Obtained. Finally, the SEW-L-17 fraction having the best antioxidant activity and hepatocyte protective activity among the 20 fractions was concentrated under reduced pressure and lyophilized to obtain 68 mg.

1-4. High-speed 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 a single substance having strong antioxidant activity and hepatocellular protective activity was prepared using high-performance liquid chromatography. Separated. For analytical HPLC, a model (1260 Infinity, Agilent, USA) equipped with a Zorbax Eclipse C18 analytical column (Zorbax Eclips, 5 μm, 4.5 × 250 mm, Agilent) was used, and high-speed fractionation liquid chromatography was performed by YMC, Japan. A model (Multiple Preparative HPLC (LC-forte/R, YMC, Japan)) equipped with a lab column (Triart C18, 20 mm × 150 mm, 5 μm, YMC, Japan) was used. Analysis was performed by flowing at a flow rate of 1 to 3 ml/min under a gradient condition using distilled water, and an Agilent, 1200 DAD detector or YMC-YUV-3400 UV detector was used. As a result of pure fractionation of the compounds using absorption, compound 1 (32 mg) could be obtained at a retention time of 13.5 minutes, Fig. 1 shows SEW-EA-B2, SEW-EA-B2-F2, and This is an analytical HPLC chromatogram of SEW-L-17 and an ultraviolet absorption wavelength spectrum of Compound 1.

Example 2: Structural analysis of compound 1 isolated from the extract of P.

The maximum absorption band of Compound 1 isolated in Example 1-4 and the absorption shift according to the addition of reagents were determined by dissolving each sample in methanol at a concentration of 1 mg/ml, and then using an ultraviolet spectrophotometer (Genesys 10S UV-VIS spectrophotometer). , Thermo Scientific, USA) in the 190-500nm region. _{At this time, AlCl 3} , NaOH, NaOAC, etc. were used to observe the change in the ultraviolet absorption band according to the addition of the reagent.

To determine the molecular weight of the compound, positive and negative scans were performed with an electron foam ionization (ESI) mass spectrometer (LC-ESI mass spectrometer, AGILENT 1100, USA Micromass Quattro II), and high resolution MS was measured.

For nuclear magnetic resonance (NMR) analysis, compound 1 (10 mg) was completely dried _{, dissolved in DMSO-d 6} (0.5 mL), injected into a 5 mm NMR tube, and used as a zeol model (JNM-ECA 600, Jeol, Japan). Analysis was performed, 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 are 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+M-H], m/z 317.0 [M+H] (Fig. 2B)

(2) Melting point: 392℃

(3) Appearance: Yellow powder

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

(5) TLC color development: FeCl ₃ (positive), bromocresol green (negative), UV light (positive, yellowish green), anilinediphenylamine (negative), antimony (negative), dragendolph (negative)

(6) UV maximum absorption wavelength range (methanol, 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, 3-H), 3.83 (3H, s, OCH ₃ ), 6.18 (1H , d, 6-H), 6.47 (1H, d, 8-H), 6.93 (1H, d, 5′-H), 7.68 (1H, m, 6′-H), 7.74 (1H, d, 2 '-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 (150MHz, DMSO-d ₆ ) δppm: 148.81(2-C), 135.86(3-C), 175.90(4-C), 156.17(5-C), 8.21(6-C), 163.94(7C) , 93.60(8-C), 160.94(9-C), 103.05(10-C), 122.00(1'-C), 111.65(2'-C), 146.-63(3'-C), 147.37 (4′-C), 115.55(5′-C), 121.72(6′-C), 55.73 (OCH3) (Fig. 2D)

(8) Chemical structural formula

Example 3: Confirmation of the inhibitory effect of non-alcoholic fatty liver induction of high-fat diet and purified salt diet of P.

3-1. Induction of obesity by feeding a high fat diet in C57BL/6J mice

36 C57BL/6J male 7-week-old mice were randomly divided into two groups as follows and fed each feed for 24 weeks to induce obesity and insulin resistance. 24 weeks in the low fat diet group (LFD, D12450B Rodent Diet with 10 kcal% fat; Research Diets, n=5) and the high fat diet group (HFD, D12492 Rodent Diet with 60 kcal% fat; Research Diets, n=31) When LFD and HFD were ingested and the average weight was compared, the body weight of the HFD intake group (52.4 ± 0.5g) was significantly higher than that of the LFD ingestion group (40.5 ± 1.8g) ( P <0.05) (Fig. 3). ).

Since it was confirmed that obesity was well induced by feeding on a high fat diet, a non-alcoholic fatty liver induction experiment was conducted using a high fat diet and a purified salt diet in obese mice that were fed on a high fat diet.

3-2. Evaluation of Efficacy in Improving Liver Damage Induced by High Fat Diet and Purified Salt Intake

The high-fat diet was D12492 (Rodent Diet with 60 kcal% fat) purchased from Research Diets in the United States, and refined salt was purchased from Hanju Geum Co., Ltd. in Korea. The salt concentration setting was calculated based on the adult daily intake (sodium 2g or salt 5g) recommended by WHO, and calculated by converting it to the rat volume ratio according to the prior literature, and the salt content is about 1% of the rat feed is recommended per day for adults. When the salt intake is the same as the amount of salt intake and calculated based on the NaCl content, respectively, the hot water extract (NaCl content: about 56.41%) prepared in 1-1 is about 1.77% of the high fat diet, and the salt intake (refined salt, NaCl content). :99.52%) is about 1.00% of the high fat diet, and based on this, feed was prepared and the experiment was conducted.

In addition, according to the prior literature that when fed a high-fat diet for a total of 36 weeks, fatty liver inflammation was clearly induced, a high-fat diet was administered for 24 weeks, followed by an additional administration of a high-fat and salt-mixed diet for a total of 37 weeks. Proceeded. After autopsy, organ weight analysis showed that the high-fat diet and purified salt (HFD + purified salt PS) group had significantly increased liver weight compared to the high-fat diet-only group. In the group that consumed the extract (HFD + Salicornia salt, SS), the weight of the liver was significantly lower (P <0.05) than the group that consumed the high fat diet and refined salt (P <0.05). (Figures 4A and 4B).

In addition, plasma obtained after autopsy was analyzed to determine the level of ALT (alanine aminotransferase), an enzyme released into the blood when hepatocytes are damaged. Compared to that, the ALT level was significantly increased, and the ALT level was significantly lower ( P <0.05) in the group that consumed the high fat diet and the extract of P. Figure 4C).

3-3. Histological evaluation of the efficacy for improving non-alcoholic fatty liver disease (NAFLD) of S.

H&E staining and Masson's trichrome staining were performed on the liver tissue obtained after autopsy, and fatty liver disease was analyzed in order to confirm the histologically the efficacy of the extract of P. oleifera extract to improve non-alcoholic fatty liver disease.

As shown in FIG. 5, fatty liver disease was suppressed in the group (HFD + Salicornia salt, SS) ingested with a high-fat diet and the Salicornia salt extract (HFD + purified salt PS) compared to the group ingested with a high-fat diet and purified salt. Showed efficacy. In other words, in the steatosis scoring category based on H&E staining, the group that consumed the high fat diet and the Salicornia salt (SS) was compared to the group that consumed the high fat diet and the purified salt (HFD + purified salt PS). It was confirmed that it inhibited fatty liver disease (P <0.094), and it was also confirmed that intrahepatic inflammation (inflammation) was also significantly suppressed.

In addition, when scoring the efficacy of improving NAFLD (non-alcoholic fatty liver disease) by synthesizing Steatosis, Inflammation, and Ballooning, it was found that the extract of Tungweed has the effect of suppressing non-alcoholic steatosis triggered by a high-fat diet and a refined salt diet. I could confirm.

For reference, the scoring criteria are as follows.

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

-Inflammation progression score in liver lobules: 0 = when there are no lesions observed under a 200-fold field microscope, 1 = when there are 2 or less lesions observed under a 200-fold field microscope, 2 = 2 lesions observed under a 200-fold field microscope When between dogs and 4, when there are 4 or more lesions observed under a 3=200× field microscope

3-4. Evaluation of the Effect of Sprout Extracts on the mRNA Expression of Inflammation and Fibrosis Markers in Liver Tissue

The mRNA expression of inflammation and hepatic fibrosis markers (Ilb, Cxcl2, Ccl2, Cd68, Col1a1) in tissue was measured as follows. RNA was extracted from the liver tissue obtained after autopsy using an RNA isolation kit (Ambion, RNA isolation kit, Life Technologies, Gaithersburg, MD, USA), and this was used in NanoDrop ND-2000 (Thermo Fisher Scientific, Waltham, MA, USA). After quantification, ^{cDNA was synthesized using a 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 2 µl of c-DNA synthesized with SYBR Green Master Mix (BioRad, Hercules, CA, USA) primers.

In PCR, primers were denatured at 95°C for 3 minutes before the amplification reaction, and were continuously reacted 44 times in 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 by CFX Connect™ Real-Time PCR Detection System (BioRad, Hercules, CA, 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′)

As shown in Figure 6, compared to the group (HFD + purified salt PS) ingested high-fat diet and purified salt (HFD + Salicornia salt, SS) compared to the group ingested high-fat diet and purified salt (HFD + Salicornia salt, SS) was an inflammation-related marker ( cxcl2). , il1b ) and fibrosis-related markers ( col1a1 ) were significantly inhibited.

Example 4: Confirmation of the efficacy of isoramnetin

4-1. Antioxidant activity investigation

In order to confirm the antioxidant activity of isoramnetin, isolated and identified as an active ingredient from the extract of Salicornia chinensis of Example 2, the scavenging ability, active oxygen scavenging ability, and lipid peroxidation inhibitory ability for DPPH free radicals were evaluated. At this time, as controls, alpha-tocopherol (vitamin E), an antioxidant vitamin used in the prevention and treatment of non-alcoholic fatty liver, and N-acetylcystein (NAC), a precursor of hepatocyte glutathione, were used.

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

Antioxidant activity was 1,1-diphenyl-2-picryl hydrazyl (1,1-) according to the method of Bullois (Chen, et. al., 1999. J. Agric. Food Chem. 47. 2226-2228). diphenyl-2-picryl hydrazyl, DPPH, Sigma Co., USA). That is, after making a DPPH solution by dissolving 5 mg of DPPH in 50 ml of ethanol, 180 μl was added to a 96-well microplate, and the sample was added at a concentration of 0-100 μg/ml, mixed for 5 seconds, and then at room temperature for 30 minutes. The reaction was carried out, and the decrease in absorbance for the control group to which the sample was not added at 517 nm was expressed as free radical scavenging activity (%). The concentration of the substance required to remove 50% of free radicals _{can be expressed as an IC 50} value, and the lower this value, the stronger the antioxidant activity.

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

As shown in Table 1, it was confirmed that the isoramnetin isolated from the lump has a free radical scavenging ability that is about 4 times stronger than alpha-tocopherol and about 1.87 times stronger than N-acetylcysteine.

4-1-2: Active oxygen scavenging ability measurement

The measurement of superoxide anion scavenging ability was carried out by using an active oxygen generator by the enzyme reaction of xanthin/xanthin oxidase, and light by oxidation of nitroblue tetrazolim (NBT) by active oxygen. It was measured using a change in absorbance (530 nm), and the concentration of a substance required to remove 50% of active oxygen is shown in _{Table 2 as an IC 50 value.}

compound Active oxygen scavenging activity (IC ₅₀ value, μM) Isoramnetine 5.3 Alpha-tocopherol >50 N-acetylcysteine 18.8

As shown in Table 2, it was confirmed that the isoramnetin isolated from the soybean sprout had an active oxygen scavenging ability that was about 9 times stronger than that of alpha-tocopherol and about 3.5 times that of N-acetylcysteine.

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

Lipid peroxidation inhibitory ability was measured using microsomes obtained by ultracentrifugation (77000g, 60 minutes, Hitachi RP 30) of extracted rat hepatocytes as a lipid source ^{to induce lipid peroxidation by Fe 2+} ascorbate system and produced malondi. By reacting aldehyde (malonaldehyde, MDA) with thiobarbituric acid (TBA), the amount of MDA reduced by the sample was quantitatively converted. 200 µl of liver microsome solution containing the sample and 225 µl of sodium dodesyl sulfate (SDS) solution were added to the test tube, and after shaking and mixing for 5 seconds, 20% acetic acid, 75 µl distilled water, 1.2% TBA 1 ml of the solution was added and warmed for 30 minutes. After cooling at room temperature for 30 minutes, centrifugation at 3000 rpm for 20 minutes and measuring the absorbance of the supernatant at 532 nm to determine the lipid peroxidation inhibitory activity (%) for the control without adding a sample, and to remove 50% of active oxygen. The concentration of was expressed as an IC ₅₀ value.

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

As shown in Table 3, it was confirmed that the isoramnetin isolated from the soybean sprout had a lipid peroxidation inhibitory ability that was about 2.2 times stronger than that of alpha-tocopherol and about 1.65 times that of N-acetylcysteine.

4-2. In LPS-stimulated Human HepG2 Cells, Isoramnetin Active Ingredients Isolated from Sprouts, Concentration-dependent NO Emission Inhibition and iNOS Protein Expression Inhibition

In order to analyze the anti-inflammatory efficacy of isoramnetin isolated from the soybean sprout in hepatocytes, nitrite (NO) produced in human HepG2 cells stimulated with LPS (100ng/ml), a hepatocyte inflammation-inducing factor, is dependent on the concentration of isoramnetin. It was confirmed that it was inhibited by. Nitrite (NO) was measured using a NO assay using Griess reagent.

As a result, as shown in FIG. 7A, nitrite (NO) induced by LPS in HepG2 cells was 25.13 ± 0.75 μM, which was an increase of about 7.5 times compared to 3.35 ± 0.52 μM used as a control. As a result of treatment at each concentration (10, 25, 50 μM), it was confirmed that the amount of nitrite (NO) decreased in a concentration-dependent manner to 20.59 ± 0.68 μM, 12.08 ± 0.46 μM, and 5.01 ± 0.09 μM, respectively.

In addition, an Immunoblotting experiment was conducted to confirm that isoramnetin inhibits the protein expression of iNOS, an enzyme that induces the synthesis of nitrite (NO) in a concentration-dependent manner. That is, in order to analyze the expression pattern of iNOS protein, isoramnetin and LPS were simultaneously treated with HepG2 cells and cultured for 18 hours, then the cells were lysed and the protein was quantified (Bradford assay, BioRad Laborotories, Hercules, CA). Next, the same amount (20 μg) of protein was loaded onto a 12% SDS-PAGE gel (Min Bio-Rad, Hets, UK), and electrophoresis was performed. The isolated protein was transferred to a cellulose membrane (nitrocelluose membrane, Hybond-C Extra, Amersham), a primary antibody against iNOS (Santa Cruz Biotechnology, Santa Cruz, CA) was combined, and then a secondary antibody (HRP-conjugated goat). ant-rabbit immunoglobulin G antibody, Sigma) was added. iNOS protein expression was confirmed using a chemiluminescence detection kit (Amersham Bioscience) with β-actin.

As shown in Figure 7B, it was confirmed that the iNOS protein amplified in the cells by LPS treatment was decreased in a concentration-dependent manner by the isoramnetin treatment, and in particular, 50 μM isoramnetin expressed the iNOS protein amplified by LPS before the LPS treatment. It was confirmed that it was suppressed to the same level.

4-3. Inhibition of the expression of NF-kB and COX-2 proteins by isoramnetin components isolated from spallia in human HepG2 cells

Expression of COX-2 and NF-kB proteins overexpressed in human HepG2 cells stimulated with LPS (100 ng/ml), a hepatocellular inflammatory factor, to analyze the inhibitory effect of isoramnetin on hepatitis triggered by non-alcoholic fatty liver. It was confirmed whether it was inhibited by this isoramnetin. COX-2 (cyclooxygenase type 2), an enzyme involved in inflammation production, is an enzyme that causes Prostaglandin E ₂ , a substance that induces inflammation in cells, and COX-2 protein expression is used as a biomarker of a typical inflammatory factor.

Because COX-2 enzyme is increased when cells are stimulated with a substance such as LPS, that is, when oxidative stress is applied within the cell or a substance such as LPS is stimulated, LPS (100 ng/ml) and LPS (100 ng/ml) in HepG2 cells and Isoramnetin was treated by concentration and incubated for 18 hours. After culturing, the protein of each cell was quantified, and Western blot was performed to confirm the expression pattern of the COX-2 protein, and the results are shown in FIG. 8A.

In the COX-2 gene promoter region, there is a site to which p65, an NFkB fragment phosphorylated by activation, binds, and it has been known that the synthesis of the COX-2 gene due to oxidative stress or inflammatory stimulation occurs due to NF-kB activation. Therefore, to confirm NFkB activated by LPS treatment through the amount of phospho-p65 protein expression in the nucleus, and to confirm whether isoramnetin also inhibits the activation of NFkB, hepG2 cells were treated with LPS (100ng/ml) and isoramnetin by concentration. After culturing for 18 hours, the nuclei of each cell were isolated, and the amount of 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, CA, USA) was used at 4°C. After lysing the separated nuclei, the supernatant was taken by high-speed centrifugation (21,000g, 30 minutes). After protein quantification, electrophoresis was performed, and the results are shown in FIG. 8B.

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

4-4. Measurement of concentration-dependent ROS production and apoptosis inhibitory ability of isoramnetin active ingredient isolated from spallia in human HepG2 cells treated with t-BHP

As one of the representative mechanisms of non-alcoholic fatty liver disease, free radicals produced during fatty acidization caused by the accumulation of liver fat due to a high fat diet cause inflammation, or direct toxicity due to oxidative stress causes cell necrosis. Is known. Therefore, whether isoramnetin can suppress the cell death caused by treatment of t-BHP (tertiary butyl hydroperoxide), which is a powerful peroxide that directly generates ROS in HepG2 cells, while eliminating ROS generated in the cell. Experimented.

1 mM t-BHP was added equally to cells treated with isoramnetin at different concentrations and cells not pretreated with isoramnetin, and 18 hours later, the amount of ROS generated in the cells and the degree of necrosis of the cells were confirmed. The intracellular ROS content generated when t-BHP was treated with HepG2 cells cultured in a 6-well plate was converted by ROS using a DCFH (2', 7'-chlorofluorescein) fluorescent probe. , 7'-chlorofluorescein diacetate) was measured at a wavelength between 485 ~ 530nm with a fluorometer (TECAN, San Jose, CA).

Cell viability due to cell necrosis was measured using the MTT assay. That is, after culturing HepG2 cells in a 6-well, isoramnetin was treated for 18 hours by concentration, and then the medium component containing isoramnetin was removed, the cells were washed with PBS, and then 1mM of t-BHP was added. I did.

From Fig. 9A, the cell viability of HepG2 cells treated with only t-BHP decreased significantly to 20.2% ± 0.88 due to cell necrosis, but the cells in the medium to which isoramnetin was added as a pretreatment were in proportion to the concentration of isoramnetin. The survival rate gradually increased, and when 50 μM of isoramnetin was treated, the cell survival rate was nearly 100%, and the effect of inhibiting oxidative stress-induced apoptosis by isoramnetin could be confirmed.

In addition, in HepG2 cells treated with only t-BHP, ROS increased rapidly, but in cells treated with isoramnetin, the production of intracellular ROS was inhibited in a concentration-dependent manner. It could be confirmed that it was lowered. The picture of FIG. 9B is a fluorescence microscope image of intracellular ROS.

4-5. Induction of glutathione synthase (GR, GCL) protein through activation of Nrf2 transcription factor of isoramnetin in human HepG2 cells and measurement of the ability to improve intracellular GSH content

In order to study the mechanism of hepatocyte inflammation and cytoprotective effects of isoramnetin, intracellular glutathione (GSH) and glutathione synthase (GR, GCL) and transcription that causes the synthesis of these enzyme proteins when isoramnetin is treated on cells It was attempted to confirm whether the factor Nrf2 was activated. The content of glutathione (GSH), known as a hepatocyte protective factor, has a close correlation between the protein expression levels of glutathione synthase glutathione reductase (GR) and gamma glutamylcysteine lyase (GCL), and recently glutathione and glutathione precursors Phosphorus N-acetylsystein has been used to treat fatty liver.

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

As shown in Figure 10A, isoramnetin concentration-dependently increases the intracellular glutathione (GSH) content, and in particular, when treated with a concentration of 50 μM or more, it was confirmed that glutathione increased more than twice as compared to the control group.

Therefore, to determine whether the increase of glutathione in HepG2 cells by isoramnetin is due to protein expression of glutathione synthetase GCL and GR following the activation of Nrf2 transcription factor by isoramnetin and phase II gene activation of Nrf2 downstream genes. Isoramnetin was treated at different concentrations (0, 2.5, 5, 10, 25, 50, 100 μM) for 18 hours, and then Western Blotting was performed, and the results are shown in FIG. 10B. For Westin blotting, isoramnetin-treated cells and control cells were lysed, and proteins were quantified (Bradford assay, BioRad Laborotories, Hercules, CA). Next, the same amount (20 μg) of protein was loaded onto a 12% SDS-PAGE gel (Min Bio-Rad, Hets, UK), and electrophoresis was performed. The isolated protein was transferred to a nitrocelluose membrane (Hybond-C Extra, Amersham), and the primary antibody against GR and GCL (Santa Cruz Biotechnology, Santa Cruz, CA) was combined, and then the secondary antibody (HRP- conjugated goat ant-rabbit immunoglobulin G antibody, Sigma) was added. The expression of GCL and GR proteins was confirmed using a chemiluminescence detection kit (Amersham Bioscience) with β-actin. As a result, it was confirmed that isoramnetin increased the expression of glutathione synthase GCL and GR in a concentration-dependent manner.

As shown in FIG. 10B, activation of the Nrf2 transcription factor by isoramnetin was confirmed by the expression level of the Nrf2 protein present in the nucleus. Isoramnetin was found to gradually activate the Nrf2 transcription factor through intranuclear migration from a concentration of 10 μM or higher, and the concentration-dependent Nrf2 activation of isoramnetin showed a similar tendency to the expression of GCL and GR.

Therefore, the increase of glutathione in HepG2 cells by isoramnetin occurs through the synthesis of GCL and GR enzyme proteins by activation of the Nrf2 transcription factor, which reduces oxidative stress caused by fatty acid oxidation in hepatocytes, which is effective in alleviating inflammation and treatment in fatty hepatocytes. Suggests that it may be.

As described above, specific parts of the present invention have been described in detail, and it will be apparent to those of ordinary skill in the art that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, it will be said that the substantial scope of the present invention is 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 (4)

( Salicornia spp. ) Extract containing isorhamnetin and sodium chloride (NaCl) capable of preventing or treating nonalcoholic fatty liver disease.
2. The extract of Salicornia spp. According to claim 1, wherein 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.
A food composition comprising Salamornia spp. Extract containing isorhamnetin and sodium chloride (NaCl) capable of preventing non-alcoholic fatty liver disease.
Isolametin with ability to prevent non-alcoholic fatty liver disease and Salicornia ( Salicornia ) containing sodium chloride (NaCl) SPP . ) Extract.

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