Anti-fibrotic composition for preventing or treating fatty liver or liver cirrhosis, containing jujube polysaccharides as active ingredient

Abstract

The present invention relates to an anti-fibrotic composition for preventing or treating fatty liver or liver cirrhosis, the composition containing jujube polysaccharides as an active ingredient. The jujube polysaccharide-containing composition according to the present invention can significantly inhibit the expression of fibrosis-related mRNAs (Tgf-β, Col1a1, Col4a1, Pdgfb, Mmp1, Mmp2, and Timp1) induced in LX2 hepatic stellate cells by TGF-β1, and the whole-cell expression and nucleus expression of fibrosis-related proteins (p-SMAD2, SMAD2, p-SMAD3, SMAD3, and SMAD4). In addition, the jujube polysaccharide-containing composition can significantly suppress body fat mass (fat mass (g), adiposity (% fat)) increased by a high-fat, high-fructose diet (HFHFD), and significantly reduce hepatic steatosis and inflammation. Therefore, the jujube polysaccharide-containing composition according to the present invention is expected to be advantageously used as an anti-fibrotic composition for preventing, alleviating, and treating fatty liver or liver cirrhosis.

Description

A composition for the prevention or treatment of antifibrosis, fatty liver, or liver cirrhosis containing jujube polysaccharides as an active ingredient

[0001] The present invention relates to a composition for the prevention or treatment of antifibrosis, fatty liver, or liver cirrhosis containing jujube polysaccharides as an active ingredient.

[0002] Metabolic dysfunction-associated steatotic liver disease (MASLD, formerly non-alcoholic fatty liver disease; NAFLD) is a group of diseases characterized by hepatic steatosis, which clinically and histologically includes simple steatosis and metabolic dysfunction-associated steatohepatitis (MASH). While simple steatosis exhibits a relatively gradual histological course, MASH is accompanied by pathological features such as ballooning degeneration, apoptosis and necrosis, and inflammatory infiltrates; if the disease progresses, there is a high risk of progression to liver fibrosis, including collagen accumulation, and cirrhosis. According to recent studies, immunopathological mechanisms play an important role in the progression of MASLD, and the need for immune modulation strategies is being emphasized (Cellular & Molecular Immunology, 22(2025) 1159-1177).

[0003] Currently, there are no commercially available treatments for this type of fatty liver hepatitis. Due to the lack of such treatments, medications for other metabolic syndromes—such as abdominal obesity, hyperlipidemia, and diabetes—are being used, including insulin resistance improvers, antioxidants (e.g., vitamins C and E), dyslipidemia treatments, and hepatoprotectors; however, these cannot be considered direct treatments for MASLD.

[0004] Steatosis is a collective term for diseases resulting from lipid metabolism abnormalities that are distinct from fatty liver hepatitis. Primarily, lipids accumulate in organs such as the brain, liver, and spleen, causing dementia, motor disorders, and seizures.

[0005] Meanwhile, the accumulation of extracellular matrix components following tissue damage is an essential physiological process for tissue repair. Unfortunately, chronic injury that exacerbates the damage leads to the overproduction of extracellular matrix components by fibroblasts and myofibroblasts. This causes an excessive accumulation of fibrous connective tissue, which in some cases leads to a pathological condition known as fibrosis.

[0006] Fibrosis can affect various organs such as the heart, liver, lungs, skeletal muscle, kidneys, and blood vessels. For example, fibrosis can occur in skeletal muscle tissue (dystrophic muscle disease), heart and blood vessel tissues (myocardial infarction), liver tissue (non-alcoholic fatty liver disease / cirrhosis), lung tissue (idiopathic pulmonary fibrosis), and kidney tissue (chronic kidney disease / renal fibrosis).

[0007] For example, renal fibrosis can occur due to various mechanisms, including excess matrix synthesis and contraction. Soma Meran et al. describe the fibroblast and myofibroblast-related mechanisms in renal fibrosis ("Fibroblasts and myofibroblasts in renal fibrosis" Int J Exp Pathol. 2011 Jun; 92(3): 158-167).

[0008] Hepatic fibrosis refers to the excessive accumulation of extracellular matrix proteins, including collagen, that occurs in most chronic liver diseases. Representative cells involved in liver fibrosis include hepatic stellate cells (HSCs), Kupffer cells, and endothelial cells. Hepatic stellate cells are the primary producers of extracellular matrix; when activated, they transform into a myofibroblast-like phenotype, leading to increased cell proliferation, increased production of various extracellular matrix substances including collagen (types I and III), and increased cell contractility.

[0009] Tissue fibrosis, defined as the excessive accumulation of the extracellular matrix (ECM), is a common pathological finding observed in lung diseases of various causes. In pulmonary fibrosis, lung fibroblasts transform into a myofibroblast phenotype, and the expression of myofibroblasts is upregulated and continuously expressed in areas where pulmonary fibrosis is progressing.

[0010] Meanwhile, jujube is a fruit whose efficacy has been proven to the extent that it is used as a medicinal herb. Jujube is rich in potassium, containing 310 mg of potassium, 42 mg of phosphorus, and 14 mg each of calcium and magnesium per 100 g. In particular, it contains 84 mcg of folic acid per 100 g, is rich in B vitamins such as thiamine, riboflavin, niacin, and pantothenic acid, and also contains beta-carotene, a precursor of vitamin A.

[0011] However, regarding patent literature related to the efficacy of jujubes, Korean Patent Publication No. 10-2023-0122793 disclosed an "antibacterial and antioxidant composition containing jujube seed extract as an active ingredient," and Korean Patent Publication No. 10-2022-0156164 disclosed an "antioxidant or anti-inflammatory composition containing jujube seed extract or buckwheat leaf extract."

[0012]

[0013] [National R&D projects that supported this invention]

[0014] [Project ID] 2710003914

[0015] [Project No.] RS-2024-00334577

[0016] [Ministry Name] Ministry of Science and ICT

[0017] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0018] [Research Project Name] Excellent Research - Mid-career Research (Type 1)

[0019] [Research Project Title] Intestinal Immunomodulatory Effects According to Structural Characteristics of Plant-Derived Polysaccharides and

[0020] Research on direct and indirect effects and mechanisms on systemic health

[0021] [Name of Project Performing Organization] Jeju National University

[0022] [Research Period] 2024.05.01 ~ 2027.04.30

[0023]

[0024] Therefore, the technical problem to be solved by the present invention is to provide a composition for the prevention, improvement, and treatment of antifibrosis, fatty liver, and liver cirrhosis, comprising a jujube-derived product as an active ingredient.

[0025] To solve the above-mentioned technical problem, the present invention provides a composition for the prevention, improvement, and treatment of antifibrosis, fatty liver, or liver cirrhosis comprising jujube polysaccharides as an active ingredient.

[0026] The above composition may be a pharmaceutical composition or a food composition.

[0027] Preferably, the date polysaccharide is characterized by being prepared according to a method comprising the following steps:

[0028] (S1) A step of concentrating jujube fruits using distilled water;

[0029] (S2) A step of removing protein from the obtained concentrate and then precipitating using ethanol to obtain a primary ethanol precipitate containing polysaccharides;

[0030] (S3) A step of dialyzing the obtained primary ethanol precipitate and then precipitating it using ethanol to obtain a secondary ethanol precipitate containing polysaccharides; and

[0031] (S4) Step of freeze-drying the above secondary ethanol precipitate.

[0032] The above protein removal is characterized by being performed using trichloroacetic acid.

[0033] Preferably, the jujube polysaccharide is fucose (L-Fucose, Fuc), rhamnose (L-Rhamnose, Rha), arabinose (L-Arabinose or D-Arabinose, Ara), galactose (D-Galactose, Gal), glucose (D-Glucose, Glc), xylose (D-Xylose, Xyl), mannose (D-Mannose, Man), fructose (D-Fructose, Fru), ribose (D-Ribose, Rib), galacturonic acid (D-Galacturonic acid, GalA), glucuronic acid (D-Glucuronic acid, GlcA), and mannuronic acid (D-Mannuronic acid). It is characterized by being composed of ManA).

[0034] Preferably, the composition is characterized by inhibiting fibrosis-related mRNA expression and fibrosis-related protein expression induced by TGF-β1.

[0035] In addition, the present invention provides a method for preventing or treating antifibrosis, fatty liver, or liver cirrhosis in animals other than humans using a composition containing jujube polysaccharides as an active ingredient.

[0036] As such, a composition containing jujube polysaccharides according to the present invention as an active ingredient can significantly inhibit the expression of fibrosis-related mRNA (Tgf-β, Col1a1, Col4a1, Pdgfb, Mmp1, Mmp2, Tim1) and fibrosis-related proteins (p-SMAD2, SMAD2, p-SMAD3, SMAD3, SMAD4) induced by TGF-β1 in LX2 hepatic stellate cells, both in total cell expression and in the nucleus. In addition, it can significantly inhibit increased body fat mass (fat mass (g), adiposity (% fat)) caused by a high-fat, high-fructose diet (HFHFD), and can significantly reduce hepatic steatosis and inflammation. Therefore, the jujube polysaccharide-containing composition according to the present invention is expected to be useful as a composition for the prevention, improvement, and treatment of antifibrosis, fatty liver, or liver cirrhosis.

[0037] The following drawings attached to this specification illustrate preferred embodiments of the present invention and serve to further enhance understanding of the technical concept of the present invention together with the aforementioned description; therefore, the present invention should not be interpreted as being limited only to the matters described in such drawings.

[0038] Figure 1 shows the results of the structural analysis of jujube polysaccharides.

[0039] Figure 2 shows the results of the 3D molecular structure analysis of jujube polysaccharides.

[0040] Figure 3 shows the cytotoxicity of jujube polysaccharides at different concentrations in LX2 hepatic stellate cells.

[0041] Figure 4 shows the inhibitory effect of jujube polysaccharide on the expression of TGF-β1-induced fibrosis-related mRNAs (Tgf-β, Col1a1, Col4a1, Pdgfb, Mmp1, Mmp2, Tim1) in LX2 hepatic stellate cells.

[0042] Figure 5 shows the inhibitory effect of date polysaccharides on the total intracellular expression of TGF-β1-induced fibrosis-related proteins (p-SMAD2, SMAD2, p-SMAD3, SMAD3, SMAD4) in LX2 hepatic stellate cells.

[0043] Figure 6 shows the inhibitory effect of jujube polysaccharide on the intranuclear expression of TGF-β1-induced fibrosis-related proteins (p-SMAD2, SMAD2, p-SMAD3, SMAD3, SMAD4) in LX2 hepatic stellate cells.

[0044] Figure 7 shows the inhibitory effect of jujube polysaccharides on collagen I (COL1A1) expression.

[0045] Figure 8 is an animal study design (A) to test the effect of adding jujube polysaccharides on a metabolic disorder-associated fatty liver disease (MASLD) model, and a representative body shape photograph (B).

[0046] Figure 9 shows the change in body weight of mice due to the intake of jujube polysaccharides.

[0047] Figure 10 shows the body weight, food intake, water intake, and calorie intake of mice after consuming jujube polysaccharides.

[0048] Figure 11 is a whole-body DEXA image of a mouse.

[0049] Figure 12 shows the effect of jujube polysaccharide intake on the body composition of mice.

[0050] Figure 13 shows the effect of jujube polysaccharide intake on fasting blood glucose levels.

[0051] Figure 14 shows the effect of date polysaccharide intake on blood total cholesterol (TC), glutamic oxaloacetic transaminase (GOT), and glutamic pyruvic transaminase (GPT) levels.

[0052] Figure 15 shows the effect of jujube polysaccharide intake on liver tissue morphology and pathological changes.

[0053] The present invention will be explained in more detail below.

[0054] The present invention relates to a composition for the prevention, improvement, and treatment of antifibrosis, fatty liver, or liver cirrhosis, comprising jujube polysaccharides as an active ingredient.

[0055] A composition containing jujube polysaccharides according to the present invention as an active ingredient can significantly inhibit the expression of fibrosis-related mRNA (Tgf-β, Col1a1, Col4a1, Pdgfb, Mmp1, Mmp2, Tim1) and fibrosis-related proteins (p-SMAD2, SMAD2, p-SMAD3, SMAD3, SMAD4) induced by TGF-β1 in LX2 cells, as well as the total intracellular and nuclear expression of these proteins.

[0056] In addition, a composition containing jujube polysaccharides according to the present invention as an active ingredient can significantly inhibit increased body fat mass (fat mass (g), adiposity (% fat)) due to a high-fat, high-fructose diet (HFHFD), and can significantly reduce hepatic steatosis and inflammation.

[0057] The jujube used in this invention is the fruit of the jujube tree, which belongs to the family Rhamnaceae, and is also called *jo* or *mokmil*. There are theories that its origin is Southern Europe, Eastern Asia, or Western Asia, and it is distributed in the temperate regions of Korea, China, Japan, and Southern Europe. As evidenced by various proverbs such as "like a kite caught in a jujube tree," "like a jujube stick," and "like a jujube seed," it is a fruit that has long been familiar in Korea. In particular, jujubes invariably bear fruit when they bloom, and the flowers do not easily fall off before ripening. Furthermore, the fruit is red, the color of the sheep, and carries the meaning of bearing many sons and ensuring the prosperity of descendants; therefore, it is an indispensable item on the table during weddings and ancestral rites. In addition to being used for food, it has long been regarded as a food that prevents aging and has been used for medicinal purposes.

[0058] These jujubes contain 36 different inorganic elements, including protein, fat, saponin, glucose, fructose, polysaccharides, organic acids, as well as calcium, phosphorus, magnesium, iron, and potassium.

[0059] The above jujube polysaccharide can be prepared according to a method comprising the following steps:

[0060] (S1) A step of concentrating jujube fruits using distilled water;

[0061] (S2) A step of removing protein from the obtained concentrate and then precipitating using ethanol to obtain a primary ethanol precipitate containing polysaccharides;

[0062] (S3) A step of dialyzing the obtained primary ethanol precipitate and then precipitating it using ethanol to obtain a secondary ethanol precipitate containing polysaccharides; and

[0063] (S4) Step of freeze-drying the above secondary ethanol precipitate.

[0064] The above protein removal can be performed using trichloroacetic acid.

[0065] Preferably, the jujube polysaccharide is fucose (L-Fucose, Fuc), rhamnose (L-Rhamnose, Rha), arabinose (L-Arabinose or D-Arabinose, Ara), galactose (D-Galactose, Gal), glucose (D-Glucose, Glc), xylose (D-Xylose, Xyl), mannose (D-Mannose, Man), fructose (D-Fructose, Fru), ribose (D-Ribose, Rib), galacturonic acid (D-Galacturonic acid, GalA), glucuronic acid (D-Glucuronic acid, GlcA), and mannuronic acid (D-Mannuronic acid). It is characterized by being composed of ManA).

[0066] In the present invention, "prevention" refers to any act of inhibiting or delaying antifibrosis, fatty liver, and liver cirrhosis by administering the above composition, and "treatment" refers to any act of improving or beneficially altering symptoms caused by antifibrosis, fatty liver, and liver cirrhosis by the above composition.

[0067] According to one embodiment of the present invention, the jujube polysaccharide is characterized by being included in an amount of 1 to 90 weight%, preferably 10 to 80 weight%, and more preferably 20 to 60 weight% based on the total weight of the composition. At this time, if the jujube polysaccharide is added in an amount of less than 1 weight%, it is difficult to obtain an improvement effect on anti-fibrosis, fatty liver, and liver cirrhosis, and if the jujube polysaccharide is added in an amount of more than 90 weight%, it is uneconomical because the effect is not proportional to the amount used.

[0068] The present invention relates to a pharmaceutical composition for the prevention or treatment of antifibrosis, fatty liver, or liver cirrhosis, comprising jujube polysaccharides as an active ingredient.

[0069] The pharmaceutical composition of the present invention may be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the active ingredient, and the adjuvants may include excipients, disintegrants, sweeteners, binders, coatings, leavening agents, lubricants, lubricants, or flavoring agents.

[0070] The above pharmaceutical composition may preferably be formulated into a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration.

[0071] The formulation form of the above pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops, or injectable liquids. For example, for formulation into the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, or water. Additionally, if desired or necessary, suitable binders, lubricants, disintegrants, and colorants may also be included in the mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose, or beta-lactose; corn sweeteners; natural and synthetic gums such as acacia, trackercanth, or sodium oleate; sodium stearate; magnesium stearate; sodium benzoate; sodium acetate; sodium chloride; etc. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, etc.

[0072] Acceptable pharmaceutical carriers for the composition formulated as the above liquid solution may include saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components, provided that they are sterile and biocompatible. Additionally, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets.

[0073] Furthermore, it can be preferably formulated according to the ingredients using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

[0074] The pharmaceutical composition of the present invention may be administered orally or parenterally. In the case of parenteral administration, it may be administered via intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc., and preferably orally.

[0075] Suitable dosages of the pharmaceutical composition of the present invention vary depending on factors such as the formulation method, mode of administration, age, body weight, sex, pathological condition, food, time of administration, route of administration, rate of excretion, and responsiveness, and a physician of ordinary skill can easily determine and prescribe a dosage effective for the intended treatment or prevention. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-10 g / kg.

[0076] The pharmaceutical composition of the present invention may be prepared in a unit volume form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier and / or excipient, according to a method that can be easily practiced by a person skilled in the art to which the invention belongs. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0077] In addition, the present invention relates to a health functional food composition for preventing or improving anti-fibrosis, fatty liver, and liver cirrhosis, comprising jujube polysaccharides as an active ingredient.

[0078] The above food composition may be in the form of tablets, capsules, powders, granules, liquids, pills, liquid preparations, syrups, juices, suspensions, emulsions, or drops. For example, for formulation into the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic, acceptable inert carrier such as ethanol, glycerol, or water. Additionally, if desired or necessary, suitable binders, lubricants, disintegrants, and colorants may also be included in the mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose, or beta-lactose; corn sweeteners; natural and synthetic gums such as acacia, trackercanth, or sodium oleate; sodium stearate; magnesium stearate; sodium benzoate; sodium acetate; sodium chloride; etc. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, etc. Acceptable pharmaceutical carriers for compositions formulated as liquid solutions include saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components, provided that they are sterile and biocompatible. Additionally, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets.

[0079] In addition, the food composition of the present invention may be used as a functional food or added to various foods. Examples of foods to which the composition of the present invention may be added include teas, beverages, meat, chocolate, jelly, food products, confectionery, pizza, ramen, other noodles, chewing gum, candies, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

[0080] In addition, the food composition may contain, in addition to the active ingredients, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and promoters (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0081] The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquid sticks, pills, etc.

[0082] In the present invention, the term "health functional food" refers to a food manufactured and processed using raw materials or ingredients that have functional properties useful to the human body in accordance with the Health Functional Foods Act, and means consuming it for the purpose of obtaining useful effects for health purposes, such as regulating nutrients or physiological actions on the structure and function of the human body.

[0083] The health functional food of the present invention may include conventional food additives, and unless otherwise specified, suitability as a food additive is determined in accordance with the specifications and standards for the relevant item, based on the general provisions and general test methods of the food additive code approved by the Ministry of Food and Drug Safety.

[0084] Examples of items listed in the above food additives code include chemically synthesized products such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamon acid; natural additives such as persimmon dye, licorice extract, crystalline cellulose, sorghum dye, and guar gum; and mixed preparations such as L-sodium glutamate preparations, alkaline noodle additives, preservative preparations, and tar dye preparations.

[0085] For example, a health functional food in tablet form may be produced by granulating a mixture of active ingredients with excipients, binders, disintegrants, and other additives using a conventional method, and then adding a lubricant or the like and compression molding, or by directly compression molding the mixture. Additionally, the health functional food in tablet form may contain a bittering agent or the like as needed.

[0086] Among the capsule-type health functional foods, hard capsules can be manufactured by filling a conventional hard capsule with a mixture in which the active ingredient of the present invention is mixed with additives such as excipients, and soft capsules can be manufactured by filling a capsule base such as gelatin with a mixture in which the extract is mixed with additives such as excipients. The soft capsules may contain plasticizers such as glycerin or sorbitol, coloring agents, preservatives, etc., as needed.

[0087] A health functional food in the form of a pill can be prepared by molding a mixture of the active ingredient of the present invention, excipients, binders, disintegrants, etc., using a previously known method, and if necessary, it can be coated with sucrose or other coating agents, or the surface can be coated with a substance such as starch or talc.

[0088] A health functional food in granular form can be manufactured into a granular form by a previously known method using a mixture of the active ingredient of the present invention, excipients, binders, disintegrants, etc., and may contain flavoring agents, bitter agents, etc. as needed.

[0089] In the preparation of the composition of the present invention, in addition to the active ingredients introduced in the present invention as food compositions, various flavoring agents and natural carbohydrates may be included as additional ingredients, as in food compositions commonly used, and physiologically acceptable auxiliary agents may be used, and said auxiliary agents may include excipients, sweeteners, coating agents, leavening agents, lubricants, binders, or flavoring agents.

[0090] The food composition of the present invention can be used as a functional food or added to various foods. Foods to which the composition added in the present invention can be added include chocolates, beverages, food products, snacks, noodles, chewing gum, candies, and health supplements.

[0091] The health functional food in this invention may include commonly used food additives, and the food additives are determined according to specifications and standards in accordance with the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety.

[0092] In addition, the present invention provides a method for preventing or treating antifibrosis, fatty liver, or liver cirrhosis in animals other than humans using a composition containing jujube polysaccharides as an active ingredient.

[0093] In this specification, the term “treatment” means a treatment performed for the purpose of preventing, improving, or alleviating a disease, symptom, or physiological abnormality, provided that this is limited to cases involving non-human animals and does not include diagnosis, treatment, or medical acts on humans.

[0094] As such, the composition containing jujube polysaccharides according to the present invention can significantly inhibit the expression of fibrosis-related mRNAs (Tgf-β, Col1a1, Col4a1, Pdgfb, Mmp1, Mmp2, Tim1) and fibrosis-related proteins (p-SMAD2, SMAD2, p-SMAD3, SMAD3, SMAD4) induced by TGF-β1 in LX2 hepatic stellate cells, as well as the total intracellular and nuclear expression of these proteins. Furthermore, it can significantly inhibit increased body fat mass (fat mass (g), adiposity (% fat)) caused by a high-fat, high-fructose diet (HFHFD), and can significantly reduce hepatic steatosis and inflammation. Therefore, the composition containing jujube polysaccharides according to the present invention is expected to be useful as a composition for the prevention, improvement, and treatment of anti-fibrosis, fatty liver, or liver cirrhosis.

[0095] The present invention will be described in more detail below using examples. These examples are intended solely to explain the present invention more specifically, and it is obvious to those skilled in the art that the scope of the present invention is not limited by them.

[0096]

[0097] <Example 1> Preparation of Jujube Polysaccharides

[0098] (1) Extraction of jujube polysaccharides

[0099] Dried jujube fruit powder is mixed with distilled water in a ratio of 1:30 and boiled to concentrate the liquid until the volume is reduced to 1 / 3. The concentrate is mixed with trichloroacetic acid in a ratio of 1:1 and reacted at room temperature. Proteins are removed by centrifugation (10 min, 8,000 rpm, room temperature) to obtain an ethanol precipitate containing polysaccharides (1:4, v:v, 12 hours). The polysaccharides obtained in this way are dissolved in distilled water in a ratio of 1:1.5, dialyzed using a 3,500 MWCO semipermeable membrane, and the polysaccharides are precipitated again with ethanol (1:4, v:v, 12 hours). The ethanol containing secondary polysaccharides obtained in this way is prepared as jujube polysaccharides (JP) through a freeze-drying process.

[0100]

[0101] (2) Monomer analysis of jujube polysaccharides

[0102] As a result of the monomer analysis of jujube polysaccharides, as shown in Table 1 below, fucose (L-Fucose, Fuc), rhamnose (L-Rhamnose, Rha), arabinose (L-Arabinose or D-Arabinose, Ara), galactose (Galactose, D-Galactose, Gal), glucose (Glucose, D-Glucose, Glc), xylose (Xylose, D-Xylose, Xyl), mannose (Mannose, D-Mannose, Man), fructose (Fructose, D-Fructose, Fru), ribose (Ribose, D-Ribose, Rib), galacturonic acid (Galacturonic acid, D-Galacturonic acid, GalA), glucuronic acid (Glucuronic acid, D-Glucuronic acid, GlcA), and mannuronic acid It was confirmed that it consists of acid, D-Mannuronic acid, and ManA.

[0103] MonomersRetention time, minmolar ratio, %Fuc3.610.367±0.009Rha7.719.130±0.071Ara8.143.294±0.071Gal10.369.481±0.194Glc11.944.053±0.089Xyl14.22.610±0.077Man 14.981.336±0.046Fru16.840.389±0.064Rib18.730.051±0.002GalA34.6968.707±0.499GlcA37.590.485±0.004ManA40.340.098±0.014

[0104]

[0105] (3) Analysis of molecular weight, polydispersity, and RMS of jujube polysaccharides Table 2 shows the results of the analysis of molecular weight, polydispersity, and RMS of jujube polysaccharides.

[0106] ItemsJPMolar mass (g / mol)Mn(1.19±0.01) ×10 5 Mw(3.76±0.01) ×10 5 Mz(8.96±0.02) ×10 5 PolydispersityMw / Mn3.15±0.013Mz / Mn2.38±0.023Root mean square radius, RMS (nm)Rn44.70±0.016Rw42.80±0.017Rz38.60±0.02

[0107]

[0108] Figure 1 shows the results of structural analysis of the date polysaccharide. Figure 2 shows the results of 3D molecular structure analysis of the date polysaccharide. Here, A is the structural formula of the date polysaccharide, B is the composition diagram of the date polysaccharide, C is the predicted molecular structure diagram of the date polysaccharide containing 281 units, and in D, the date polysaccharide exhibits a helical thin plate structure, and it can be confirmed through the SEM image that it also exhibits a thin plate shape.

[0109]

[0110] <Test Example 1> Cytotoxicity Test

[0111] The concentration-dependent cytotoxicity of jujube polysaccharides in LX2 hepatic stellate cells was measured using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay.

[0112] 1.25 × 10⁶ LX2 cells in a 96-well plate 4 Cells were seeded at a rate of cells / well and cultured overnight. Subsequently, jujube polysaccharides were added at various concentrations (100, 200, 400, 800, 1,000 μg / mL) and cultured for 48 hours. MTT stock solution was diluted in the cell culture medium to a final concentration of 0.2 mg / mL and incubated for 3 hours. After removing the culture medium, 50 μL of DMSO was added to each well to dissolve the formazan. The plates were shaken for 10 minutes under light-blocking conditions, and absorbance was measured at 540 nm using a microplate reader.

[0113] Figure 3 shows the cytotoxicity of jujube polysaccharides at different concentrations in LX2 hepatic stellate cells. As seen here, jujube polysaccharides did not show a significant difference compared to the control group up to a concentration of 400 μg / mL, but significant cytotoxicity was observed at concentrations of 800 and 1000 μg / mL.

[0114]

[0115] <Test Example 2> Measurement of the inhibitory effect on fibrosis-related mRNA expression

[0116] The inhibitory effect of jujube polysaccharides on the expression of fiber-related mRNAs (Tgf-β, Col1a1, Col4a1, Pdgfb, Mmp1, Mmp2, Tim1) was measured.

[0117] LX2 cells were seeded into 6-well plates and cultured overnight. After treatment with Starve media for 4 hours, JP and TGF-β1 were administered for 48 hours. After washing twice with cold PBS, RNA was extracted from LX2 cells using Trizol reagent, and all experiments were performed under RNase-free conditions. The extracted RNA was quantified using NanoDrop, and 20 μL of cDNA was synthesized using the ABI High Capacity cDNA Archive kit with a total of 2 μg of mRNA (synthesis conditions: 25°C for 10 min, 37°C for 2 hours, or 85°C for 5 min). The synthesized cDNA was diluted 5-fold with RNase-free water for use in the experiments. Target RNA expression was measured using real-time qPCR with a final reaction mixture of 10 μL, prepared by mixing 2.5 μL of cDNA, 5 μL of SYBR Green, and forward and reverse primers with a final concentration of 1.6 μM. PCR conditions were repeated 40 times under 50 ℃ / 2 min; 95 ℃ / 2 min; 95 ℃ / 15 sec; and 60 ℃ / 1 min. The final extension step was performed for 0.05 sec at 65 ℃ using a Roche Fast Start Universal SYBR Green Master. Relative gene expression was 2 -ΔΔCt Calculations were performed using the method, and the results were standardized using the ribosomal protein lateral stalk subunit p0 (Rplp0, 36b4) as a housekeeping gene (internal control).

[0118] Figure 4 shows the inhibitory effect of jujube polysaccharide on the expression of TGF-β1-induced fibrosis-related mRNA (Tgf-β, Col1a1, Col4a1, Pdgfb, Mmp1, Mmp2, Tim1) in LX2 hepatic stellate cells. As shown here, it was confirmed that jujube polysaccharide (400 μg / mL) significantly inhibits the expression of TGF-β1-induced fibrosis-related mRNA (Tgf-β, Col1a1, Col4a1, Pdgfb, Mmp1, Mmp2, Tim1) in LX2 cells.

[0119]

[0120] <Test Example 3> Measurement of inhibitory effect on fibrosis-related protein expression (whole cells)

[0121] The inhibitory effect of jujube polysaccharides on the expression of fibrosis-related proteins (p-SMAD2, SMAD2, p-SMAD3, SMAD3, SMAD4) in whole cell lysate was measured.

[0122] LX2 cells were seeded into 6-well plates and cultured overnight. Subsequently, starve media was added for 4 hours. After adding JP and TGFβ and culturing for 48 hours, RIPA buffer was added to the LX2 cells to extract proteins, and the mixture was incubated on ice for 60 minutes. The supernatant was then obtained by centrifugation. Protein concentration was measured using the BCA protein quantification method. The prepared protein samples were mixed with RIPA buffer and 4X buffer containing 10% dithiothreitol (DTT) to a final concentration of 30 μg. The prepared proteins were subjected to electrophoresis on a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) at 80 V for 2 hours. After electrophoresis, the proteins contained in the gel were transferred to a polyvinylidene difluoride (PVDF) membrane, and the membrane was blocked for 1 hour at room temperature using 5% skim milk prepared with Tris-buffered saline (TBS+). Subsequently, the membrane was washed three times with TBS for 10 minutes each, and the primary antibody, diluted at a ratio of 1:1000 in TBS containing 3% BSA, was added and incubated overnight at 4°C. The next day, the membrane was washed three times, and the secondary antibody, diluted at a ratio of 1:3000 in TBS containing 3% BSA, was incubated for 1 hour at room temperature. After washing the membrane conjugated with the secondary antibody with TBS, the expressed protein was detected using an ECL kit. Protein expression was confirmed and analyzed using a ChemiDoc instrument.

[0123] Figure 5 shows the inhibitory effect of date polysaccharide on the total intracellular expression of TGF-β1-induced fibrosis-related proteins (p-SMAD2, SMAD2, p-SMAD3, SMAD3, SMAD4) in LX2 hepatic stellate cells. As shown here, date polysaccharide significantly inhibited the total intracellular expression of TGF-β1-induced fibrosis-related proteins (p-SMAD2, SMAD2, p-SMAD3, SMAD3, SMAD4) in LX2 cells.

[0124]

[0125] <Test Example 4> Measurement of inhibitory effect on fibrosis-related protein expression (cell nucleus)

[0126] The inhibitory effect of jujube polysaccharides on the expression of fibrillation-related proteins (p-SMAD2, SMAD2, p-SMAD3, SMAD3, SMAD4) in the nucleus fraction was measured.

[0127] LX2 cells were seeded into 6-well plates and cultured overnight. Subsequently, they were treated with starve meida for 4 hours. After adding JP and TGFβ and culturing for 48 hours, the LX2 cells were fractionated into nuclei and cytoplasm using the NE-PER nuclear / cytoplasmic extraction kit. Western blot analysis was performed using GAPDH and histone H3 as loading controls for the cytoplasm and nuclei, respectively. A total of 20 μg of protein was subjected to electrophoresis on a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) at 80V for 2 hours. Following electrophoresis, the proteins contained in the gel were transferred to a polyvinylidene difluoride (PVDF) membrane at 300mA for 1 hour and 30 minutes. The transferred membranes were blocked at room temperature for 1 hour using 5% skim milk prepared with TBS containing Tween 20. After washing the membrane three times for 10 minutes each, the primary antibody was diluted 1:1,000 in TBS containing 3% BSA and incubated overnight at 4°C. The next day, the membrane was washed three times for 10 minutes each with TBS, and the secondary antibody was diluted 1:3,000 in TBS containing 3% BSA and incubated for 1 hour at room temperature. After washing the membrane conjugated with the secondary antibody with TBS, the expressed protein was detected using an ECL kit. Protein expression was confirmed and analyzed using a ChemiDoc instrument.

[0128] Figure 6 shows the inhibitory effect of jujube polysaccharide on the nuclear expression of TGF-β1-induced fibrosis-related proteins (p-SMAD2, SMAD2, p-SMAD3, SMAD3, SMAD4) in LX2 hepatic stellate cells. As shown here, jujube polysaccharide significantly inhibited the nuclear expression of TGF-β1-induced fibrosis-related proteins (p-SMAD2, SMAD2, p-SMAD3, SMAD3, SMAD4) in LX2 cells.

[0129]

[0130] <Test Example 4> Measurement of Collagen I (COL1A1) Expression Inhibitory Effect

[0131] The inhibitory effect of jujube polysaccharides on collagen I (COL1A1) expression was measured through immunofluorescence staining.

[0132] LX2 cells were seeded onto 12 mm cover slips and cultured overnight, followed by treatment with starve meida for 4 hours. Subsequently, JP and TGF-β1 were added, and the cells were cultured for 48 hours. After culture, the supernatant was removed, and the cells were fixed with 10% formalin for 10 minutes, followed by washing three times with phosphate buffer saline (PBS). The fixed cells were treated with 0.25% Triton X-100 solution for 10 minutes to induce permeability, followed by washing three more times with PBS. To prevent non-specific antibody binding, the cells were incubated in 5% bovine serum albumin (BSA) solution for 30 minutes. Afterward, the fixed cells were immersed in a blocking solution containing the COL1A1 primary antibody and cultured at 4°C for 12 hours. After removing the primary antibody solution and washing with PBS, the cells were incubated in a secondary antibody solution containing Alexa Fluor 488 in the dark for 1 hour. After incubation, the cells were washed with PBS and mounted in a mounting solution containing DAPI (4',6-Diamidino-2-Phenylindole, dihydrochloride). Intracellular COL1A1 expression was observed using a confocal microscope, and COL1A1 expression was quantified using the image analysis program Image J.

[0133] Figure 7 shows the inhibitory effect of jujube polysaccharides on collagen I (COL1A1) expression. As shown here, jujube polysaccharides inhibited collagen I (COL1A1) expression.

[0134]

[0135] <Test Example 5> Measurement of changes following the intake of jujube polysaccharides

[0136] (1) Design of an animal study for a metabolic disorder-associated fatty liver disease (MASLD) model

[0137] The effect of adding 5% jujube polysaccharide on a mouse model of metabolic disorder-associated fatty liver disease (MASLD) induced by a high-fat, high-fructose diet (HFHFD) was confirmed.

[0138] 5-week-old male C57BL / 6J mice were used, and the mice were divided into normal diet (ND), normal diet + 5% date polysaccharide (N / JP), high-fat, high-fructose diet (HFHFD), and high-fat, high-fructose diet + 5% date polysaccharide (H / JP). The experiment was conducted for a total of 17 weeks, and results were evaluated through fasting blood glucose and DEXA analysis before the end of the experiment.

[0139] Figure 8 is an animal study design (A) to test the effect of adding jujube polysaccharides on a metabolic disorder-associated fatty liver disease (MASLD) model, and a representative body shape photograph (B).

[0140]

[0141] (2) Measurement of the effect of jujube polysaccharide intake on mice with metabolic disorder-associated fatty liver disease (MASLD)

[0142] Figure 9 shows the changes in body weight of mice due to the intake of jujube polysaccharides. As seen here, the body weight of the group supplemented with jujube polysaccharide JP to the normal diet (ND) decreased significantly compared to the ND group between weeks 10 and 13. However, the group supplemented with jujube polysaccharide JP to the high-fat, high-fructose diet (HFHFD) did not show a significant difference compared to the HFHFD group.

[0143] Figure 10 shows the body weight, food intake, water intake, and caloric intake of mice after consuming jujube polysaccharides. As can be seen here, H / JP showed no significant difference in food intake compared to HFHFD, but water intake and caloric intake were significantly reduced.

[0144] Figure 11 is a whole-body DEXA image of mice. Through these DEXA images, the body composition of mice in each group can be visually confirmed. The JP-added group shows a tendency for reduced fat accumulation.

[0145] Figure 12 shows the effects of jujube polysaccharide intake on body composition in mice. As shown here, JP significantly inhibited the increased body fat mass (fat mass (g), adiposity (% fat)) caused by HFHFD. In addition, a protective effect of JP was observed in bone density indicators.

[0146]

[0147] (3) Measurement of blood biochemical changes in mice with metabolic disorder-associated fatty liver disease (MASLD) after intake of jujube polysaccharides

[0148] Fasting blood glucose was measured using a blood glucose meter after fasting mice at week 14 and collecting blood from their tails.

[0149] Blood was collected from the hearts of experimental animals after fasting for 12 hours at the end of the experiment. The collected blood was centrifuged to separate the plasma, which was stored at -80°C for analysis. For the biochemical analysis of the blood, total cholesterol (TC), glutamic oxaloacetic transaminase (GOT), and glutamic pyruvic transaminase (GPT) were measured using an enzyme-based quantitative assay kit.

[0150] Figure 13 shows the effect of jujube polysaccharide intake on fasting blood glucose levels. As seen here, the N / JP group showed significantly lower levels compared to the ND group. However, no significant difference was observed between the H / JP group and the HFHFD group.

[0151] Figure 14 shows the effects of jujube polysaccharide intake on blood total cholesterol (TC), GOT, and GPT levels. As seen here, blood total cholesterol (TC) and GOT levels were significantly reduced in the H / JP group compared to the HFHFD group, but the effect of JP addition on GPT levels was not statistically significant.

[0152]

[0153] <Test Example 6> Effect of Addition of Jujube Polysaccharide on Liver Morphology, Pathological Sections, and Hepatopathology Score in Mice with Metabolic Disorder-Associated Fatty Liver Disease (MASLD)

[0154] The effects of adding jujube polysaccharides on liver morphology, pathological sections, and hepatopathology scores in mice with metabolic disorder-associated fatty liver disease (MASLD) were measured.

[0155] Mouse liver tissue was fixed in a 10% neutral formalin solution for 24 hours, followed by dehydration and paraffin embedding to prepare blocks. The prepared paraffin blocks were sectioned to a thickness of 3.5 μm using a microtome, and the sections were attached to slides and dried to fix the tissue. The slides were immersed in xylene solution three times for 5 minutes each to remove paraffin, and then rehydrated by dehydrating them in 100%, 100%, 95%, 90%, 80%, and 70% ethanol for 3 minutes each, in that order. Subsequently, hematoxylin and eosin (H&E) staining was performed, and the stained tissues were observed and photographed under a light microscope.

[0156] Figure 15 shows the effect of jujube polysaccharide intake on fasting blood glucose levels. Here, A and B represent the pre- and post-operative morphology of the liver. CH represents the liver pathological section. F represents steatosis. G represents inflammation. H represents liver injury cells. As shown in Figure 15, the livers of the HFHFD group were larger and lighter in color when observed visually from the anterior and posterior views compared to the ND group, and nodules due to fat accumulation appeared on the surface. Additionally, the H / JP group alleviated these liver phenomena.

[0157] In addition, the morphology of liver sections was observed at magnifications of 10x, 20x, and 40x in the H&E-stained liver pathology sections of CH, and grading was performed based on this (CE). As shown here, it was confirmed that hepatic steatosis and inflammation caused by HFHFD were significantly reduced in the group treated with JP (F, G). Furthermore, JP showed a tendency to alleviate liver cell injury (p=0.059), but this was not statistically significant (H).

[0158] The following exemplifies a pharmaceutical formulation using the jujube polysaccharide of the present invention.

[0159]

[0160] <Preparation Example 1> Preparation of a pharmaceutical formulation

[0161] <1-1> Preparation of Injectables

[0162] Jujube polysaccharide 300 mg

[0163] Sodium metabisulfite 3.0 mg

[0164] Methylparaben 0.8 mg

[0165] Propylparaben 0.1 mg

[0166] Appropriate amount of sterile distilled water for injection

[0167] The above ingredients were mixed and prepared by a conventional method to a final volume of 2 ml, filled into an ampoule, and sterilized to produce an injectable drug.

[0168]

[0169] <1-2> Preparation of Powders

[0170] Jujube polysaccharide 10 mg

[0171] 1 g lactose

[0172] The above ingredients were mixed and filled into an airtight bag to manufacture a powder.

[0173]

[0174] <1-3> Preparation of Tablets

[0175] Jujube polysaccharide 0.1 mg

[0176] 100 mg of corn starch

[0177] 100 mg lactose

[0178] Magnesium stearate 2 mg

[0179] After mixing the above ingredients, tablets were manufactured by compressing them according to the conventional method of manufacturing tablets.

[0180]

[0181] <1-4> Preparation of Capsules

[0182] Jujube polysaccharide 0.1 mg

[0183] 100 mg of corn starch

[0184] 100 mg lactose

[0185] Magnesium stearate 2 mg

[0186] After mixing the above ingredients, a capsule was manufactured by filling it into a gelatin capsule according to a conventional method for manufacturing capsules.

[0187]

[0188] <1-5> Preparation of pills

[0189] Jujube polysaccharide 1 mg

[0190] 1.5 g lactose

[0191] 1 g glycerin

[0192] 0.5 g xylitol

[0193] After mixing the above ingredients, the mixture was prepared according to a conventional method so that each pill weighed 4 g.

[0194]

[0195] <1-6> Preparation of Granules

[0196] Jujube polysaccharide 0.15 mg

[0197] Soybean extract 50 mg

[0198] 200 mg of glucose

[0199] 600 mg of starch

[0200] After mixing the above ingredients, 100 mg of 30% ethanol was added and dried at 60°C to form granules, which were then filled into a bag.

[0201]

[0202] The following exemplifies a formulation of a health drink using the jujube polysaccharide of the present invention.

[0203] <Preparation Example 2> Preparation of a health drink

[0204] <2-1> Preparation of Health Drinks

[0205] 0.5 g of the jujube polysaccharide of the present invention was homogeneously mixed with auxiliary ingredients such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%), and water (75%), and then instantaneously sterilized and packaged in small packaging containers such as glass bottles and PET bottles.

[0206]

[0207] <2-2> Preparation of Vegetable Juice

[0208] Vegetable juice was prepared by adding 0.5 g of the jujube polysaccharide of the present invention to 1,000 mL of tomato or carrot juice.

[0209]

[0210] <2-3> Preparation of Fruit Juice

[0211] Fruit juice was prepared by adding 0.1 g of the jujube polysaccharide of the present invention to 1,000 mL of apple or grape juice.

[0212] Although the above composition ratio was formulated by mixing ingredients relatively suitable for beverages as a preferred embodiment, the mixing ratio may be arbitrarily modified according to regional and ethnic preferences, such as consumer groups, countries of demand, and intended uses.

[0213] The following exemplifies a health food formulation using the jujube polysaccharide of the present invention.

[0214]

[0215] <Preparation Example 3> Preparation of health food

[0216] Food products containing the jujube polysaccharide of the present invention as an active ingredient were prepared as follows.

[0217] <3-1> Manufacturing of Flour Foods

[0218] 0.5 to 5.0 parts by weight of the jujube polysaccharide of the present invention was added to flour, and bread, cake, cookies, crackers, and noodles were manufactured using this mixture.

[0219]

[0220] <3-2> Preparation of Soups and Gravies

[0221] 0.1 to 5.0 parts by weight of the jujube polysaccharide of the present invention was added to soup and meat broth to produce meat processing products for health promotion, soup and meat broth for noodles.

[0222]

[0223] <3-3> Preparation of Ground Beef

[0224] Health-promoting ground beef was prepared by adding 10 parts by weight of the jujube polysaccharide of the present invention to ground beef.

[0225]

[0226] <3-4> Manufacturing of Dairy Products

[0227] 5 to 10 parts by weight of the date polysaccharide of the present invention were added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

[0228]

[0229] <3-5> Preparation of Seonsik

[0230] Brown rice, barley, glutinous rice, and Job's tears were gelatinized and dried using a known method, then roasted and ground into a powder with a particle size of 60 mesh using a grinder.

[0231] Black beans, black sesame seeds, and perilla seeds were also steamed and dried using the known method, then roasted and ground into a powder with a particle size of 60 mesh using a grinder.

[0232] The jujube polysaccharide of the present invention was concentrated under reduced pressure in a vacuum concentrator and dried using a spray and hot air dryer to obtain a dried product, which was then ground to a particle size of 60 mesh using a grinder to obtain a dried powder.

[0233] The grains and seeds prepared above and the jujube polysaccharide of the present invention were mixed in the following proportions to produce the product.

[0234] Grains (30 parts by weight of brown rice, 15 parts by weight of Job's tears, 20 parts by weight of barley),

[0235] Seeds (7 parts by weight perilla, 8 parts by weight black soybeans, 7 parts by weight black sesame),

[0236] Jujube polysaccharide of the present invention (3 parts by weight),

[0237] Reishi mushroom (0.5 parts by weight),

[0238] Rehmannia glutinosa (0.5 parts by weight)

[0239]

[0240] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention shall be defined by the appended claims and their equivalents.

[0241]

[0242] As such, a composition containing jujube polysaccharides according to the present invention as an active ingredient can significantly inhibit the expression of fibrosis-related mRNAs (Tgf-β, Col1a1, Col4a1, Pdgfb, Mmp1, Mmp2, Tim1) and fibrosis-related proteins (p-SMAD2, SMAD2, p-SMAD3, SMAD3, SMAD4) induced by TGF-β1 in LX2 cells, as well as the total intracellular and nuclear expression of these proteins. Furthermore, it can significantly inhibit increased body fat mass (fat mass (g), adiposity (% fat)) caused by a high-fat, high-fructose diet (HFHFD), and can significantly reduce hepatic steatosis and inflammation. Therefore, the jujube polysaccharide-containing composition according to the present invention is expected to be useful as a composition for the prevention, improvement, and treatment of anti-fibrosis, fatty liver, or liver cirrhosis.

Claims

1. A pharmaceutical composition for the prevention or treatment of antifibrosis, fatty liver, or liver cirrhosis, characterized by containing jujube polysaccharides as an active ingredient.

2. In Paragraph 1, A pharmaceutical composition for the prevention or treatment of antifibrosis, fatty liver, or liver cirrhosis, characterized in that the above-mentioned jujube polysaccharide is prepared by a method comprising the following steps: (S1) A step of concentrating jujube fruits using distilled water; (S2) A step of removing protein from the obtained concentrate and then precipitating using ethanol to obtain a primary ethanol precipitate containing polysaccharides; (S3) A step of dialyzing the obtained primary ethanol precipitate and then precipitating it using ethanol to obtain a secondary ethanol precipitate containing polysaccharides; and (S4) Step of freeze-drying the above secondary ethanol precipitate.

3. In Paragraph 2, A pharmaceutical composition for the prevention or treatment of antifibrosis, fatty liver, or liver cirrhosis, characterized in that the protein removal in step (S2) above is performed using trichloroacetic acid.

4. In Paragraph 1, The above jujube polysaccharides are fucose (L-Fucose, Fuc), rhamnose (L-Rhamnose, Rha), arabinose (L-Arabinose or D-Arabinose, Ara), galactose (D-Galactose, Gal), glucose (D-Glucose, Glc), xylose (D-Xylose, Xyl), mannose (D-Mannose, Man), fructose (D-Fructose, Fru), ribose (D-Ribose, Rib), galacturonic acid (D-Galacturonic acid, GalA), glucuronic acid (D-Glucuronic acid, GlcA), and mannuronic acid (D-Mannuronic acid, ManA). A pharmaceutical composition for the prevention or treatment of antifibrosis, fatty liver, or liver cirrhosis, characterized by being made of the following.

5. In Paragraph 1, The above composition is a pharmaceutical composition for the prevention or treatment of antifibrosis, fatty liver, or liver cirrhosis, characterized by inhibiting fibrosis-related mRNA expression and fibrosis-related protein expression induced by TGF-β1.

6. A health food composition for the improvement or prevention of anti-fibrosis, fatty liver, or liver cirrhosis, characterized by containing jujube polysaccharides as an active ingredient.

7. In Paragraph 6, A health food composition for the improvement or prevention of antifibrosis, fatty liver, or liver cirrhosis, characterized in that the above-mentioned jujube polysaccharide is prepared by a method comprising the following steps: (S1) A step of concentrating jujube fruits using distilled water; (S2) A step of removing protein from the obtained concentrate and then precipitating using ethanol to obtain a primary ethanol precipitate containing polysaccharides; (S3) A step of dialyzing the obtained primary ethanol precipitate and then precipitating it using ethanol to obtain a secondary ethanol precipitate containing polysaccharides; and (S4) Step of freeze-drying the above secondary ethanol precipitate.

8. In Paragraph 7, A health food composition for the improvement or prevention of antifibrosis, fatty liver, or liver cirrhosis, characterized in that the protein removal in step (S2) above is performed using trichloroacetic acid.

9. In Paragraph 6, The above jujube polysaccharides are fucose (L-Fucose, Fuc), rhamnose (L-Rhamnose, Rha), arabinose (L-Arabinose or D-Arabinose, Ara), galactose (D-Galactose, Gal), glucose (D-Glucose, Glc), xylose (D-Xylose, Xyl), mannose (D-Mannose, Man), fructose (D-Fructose, Fru), ribose (D-Ribose, Rib), galacturonic acid (D-Galacturonic acid, GalA), glucuronic acid (D-Glucuronic acid, GlcA), and mannuronic acid (D-Mannuronic acid, ManA). A health food composition for the improvement or prevention of anti-fibrosis, fatty liver, or liver cirrhosis, characterized by being made.

10. In Paragraph 6, The above composition is a health food composition for the improvement or prevention of antifibrosis, fatty liver, or liver cirrhosis, characterized by inhibiting fibrosis-related mRNA expression and fibrosis-related protein expression induced by TGF-β1.

11. A method for preventing or treating antifibrosis, fatty liver, or liver cirrhosis in animals other than humans using a composition containing jujube polysaccharides as an active ingredient.

Images