KR20190044171A - pharmaceutical composition for the prevention or treatment of liver disease comprising a gomisine derivative as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating liver diseases containing a gomisin derivative as an active component. The gomisin derivative according to the present invention inhibits the differentiation of hepatic stellate cells, reduces accumulation of proteins of type1, A1 collagen fibers, and inhibits the expression of α-SMA and mRNA of collagen genes, thereby being useful as the pharmaceutical composition for preventing or treating liver diseases.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for prevention or treatment of liver disease,

The present invention relates to a pharmaceutical composition for preventing or treating liver disease comprising a hypermucin derivative as an active ingredient.

Liver is one of the biggest organ in our body. It produces protein, sugar, vitamin, fat, bile, which helps absorption of fat and fat soluble vitamin. Alcohol, ammonia and various drugs are harmful to body. It acts to detoxify toxic substances. Liver fibrosis is an early process that progresses from liver cirrhosis to liver cirrhosis, resulting in excessive accumulation of collagen in the liver (J Biol Chem, 275 (2000), pp. 2247-2250). If liver fibrosis persists chronicly, it develops into cirrhosis / cirrhosis, impairing liver function, destroying the liver's structural structure, and eventually progressing to liver cancer. Chronic liver disease is an important adult disease in Korea, which is characterized by appetite decline and chronic fatigue symptoms. It is cirrhotic in more than 80% of liver cancer, the second highest cancer death rate in Korea after lung cancer in 2013.

Liver fibrosis is caused by a wound healing action against chronic stimuli such as viral hepatitis, alcoholic hepatitis, alcohol poisoning or toxic drugs. When hepatocytes are damaged by various causes, Kupffer cells, which are a kind of phagocytic cells, are transformed with TGF-β, platelet-derived growth factor (PDGF), tumor necrosis factor-α TNF-α), and regenerates damaged liver tissue by inducing inflammation [Alcoholism Clin Exp Res 1999; 23: 911-6]. Hepatic stellate cells (HSCs) located between hepatocytes and sinusoidal endothelial cells are composed of active substances secreted from Cooper and damaged hepatocytes, (J Gastroenterol Hepatol, 14 (1999), pp. 618-633), an extracellular interstitial substance such as proteoglycan, including collagen fibers (ECM ) To promote tissue regeneration by depositing new extracellular stromal materials around the damaged liver tissue (J Gastroenterol 2000; 35: 665-672).

However, when hepatocyte destruction and regeneration occurs repeatedly, the collagen proteins of the extracellular matrix in all causes of liver damage are transformed from a fine fibril state to a fibrous state and accumulate, resulting in hepatic fibrosis ) Process (Semin Liver Dis 1990; 10: 30-46). The most commonly observed features of liver fibrosis are dramatic increases in type 1 collagen fibers and a small but significant increase in type 3 collagen fibers. In particular, type 1 collagen fibers account for about 2% of total protein in normal liver tissue, but it is known to increase to 10 ~ 30% of total protein in hepatic fibrotic liver tissue (Conn Tis Res 1989 23: 19-31). Thus, when large amounts of collagen fibers accumulate and liver fibrosis persists, the liver structure changes, liver function decreases, and liver cirrhosis progresses to liver stiffness.

Liver cirrhosis / cirrhosis is irreversible, but liver fibrosis is a reversible reaction, so prevention and early treatment are possible. Therefore, in order to effectively prevent and treat cirrhosis / cirrhosis, it is absolutely important to inhibit the activity of hepatic stellate cells (HSC) to block the progress of liver fibrosis by blocking the synthesis of collagen fibers and the like (J Gastroenterol 2000; 35: 665-672).

Accordingly, the present inventors have found that the effect of Gomisin G and / or Gomisin O inhibiting the differentiation of hepatic stellate cells and reducing the protein accumulation of type I A1 collagen fibers and the mRNA expression of the collagen gene And the present invention has been completed.

Korean Patent Laid-Open No. 10-2009-0069720

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating liver disease comprising at least one of Gomisin G and Gomisin O.

Another object of the present invention is to provide a health functional food composition for preventing or ameliorating liver disease comprising at least one of Gomisin G and Gomisin O.

It is still another object of the present invention to provide a health food composition for preventing or ameliorating liver diseases comprising at least one of Gomisin G and Gomisin O.

In order to achieve the above object,

The present invention provides a pharmaceutical composition for preventing or treating liver disease comprising at least one of Gomisin G and Gomisin O.

The present invention also provides a health functional food composition for preventing or ameliorating liver disease comprising at least one of Gomisin G and Gomisin O.

Further, the present invention provides a health functional food composition for preventing or ameliorating liver diseases comprising at least one of Gomisin G and Gomisin O.

The hypermucin derivatives according to the present invention have an effect of inhibiting the differentiation of hepatic stellate cells, decreasing protein accumulation of type I A1 collagen fibers, inhibiting the expression of mRNA of? -SMA and collagen gene, May be useful as pharmaceutical compositions.

FIG. 1 is a diagram illustrating an experimental procedure for inhibiting liver fibrosis of Schizandra chinensis extract in a mouse liver injury model by TAA.
FIG. 2 shows the results of experiments in which ALT and AST enzyme activities were assayed for the liver function recovery effect of Omija extract administered to mice induced by TAA.
FIG. 3 shows the results of observing the inhibitory effect of Schizandra chinensis extract on liver fibrosis by tissue staining in mice induced by hepatic injury by TAA.
FIG. 4 shows the results of decreasing the amount of α-SMA and type 1 A1 collagen fibers (COL1A1) expressed in mouse liver tissue induced by liver injury by TAA by the extract of Omija
FIG. 5 shows the result of RT-PCR analysis of a decrease in the expression of α-SMA and type 1 A1 collagen (COL1A1) gene mRNA, which is increased by TGFβ, upon treatment with an extract of Omija for hepatic stellate cells (HSC) .
FIG. 6 shows the result of QRT-PCR analysis of a decrease in the expression of the type 1 A1 collagen (COL1A1) gene, which is increased by TGFβ when the HSC is treated with Omija extract.
FIG. 7 shows the results of RT-PCR analysis of an extract fraction of Schizandra chinensis, which reduces expression of α-SMA and type 1 A1 collagen (COL1A1) gene mRNA expressed by TGFβ in hepatic stellate cells (HSC).
FIG. 8 is a chromatogram of nine authentic samples included in Omija.
Figure 9 is a chromatogram of the gomisin derivatives contained in the hexane fraction of Omija.
10 shows the effect of the hypermucin derivatives isolated from the omizahexane fraction layer on the expression of α-SMA and type 1 A1 collagen (COL1A1) gene mRNA expressed by TGFβ in hepatic stellate cells (HSC) by RT-PCR It is the result of the experiment by the law.

Hereinafter, the present invention will be described in detail.

A pharmaceutical composition for prevention or treatment of liver disease comprising hypericin G and / or hypericin O as an active ingredient

The present invention provides a pharmaceutical composition for preventing or treating liver disease comprising Gomisin G and / or Gomisin O as an active ingredient.

The liver diseases for which the pharmaceutical composition of the present invention can be treated, prevented or ameliorated include, but are not limited to, liver cirrhosis, hepatic fibrosis, acute hepatitis, chronic hepatitis or liver cancer, preferably liver cirrhosis or liver fibrosis have.

In one embodiment of the present invention, the Gomisin G and / or Gomisin O can be separated from the fraction of Omiza extract and used for the extraction. Any suitable solvent that can be used in the art may be used, and water or an organic solvent may be used. Examples of the solvent include alcohols having 1 to 4 carbon atoms, acetone, ether, and the like, including purified water, methanol, ethanol, propanol, isopropanol, butanol, Various solvents such as benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane may be used alone or in combination. But is not limited to.

In one embodiment of the present invention, the fraction is characterized by fractionating the Omiza extract with butanol, hexane, chloroform or ethyl acetate, and preferably fractionation using hexane.

When the composition of the present invention is used as a medicine, the composition may be formulated into various oral or non-oral dosage forms at the time of clinical administration, but is not limited thereto.

Examples of the formulations for oral administration include tablets, pills, light / soft capsules, liquids, suspensions, emulsions, syrups, granules and elixirs. These formulations may contain, in addition to the active ingredient, a diluent (e.g., lactose, dextrose, (For example, silica, talc, stearic acid and magnesium or calcium salts thereof and / or polyethylene glycol), and the like. The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine and may optionally contain additives such as starch, agar, alginic acid or its sodium salt A disintegrating or boiling mixture and / or an absorbent, a colorant, a flavoring agent, and a sweetening agent.

In addition, the dosage of human gomisin G and / or gomisin O according to the present invention may vary depending on the patient's age, weight, sex, dosage form, health condition and disease severity And is generally 0.1 to 1,000 mg / day, preferably 1 to 500 mg / day on the basis of an adult patient weighing 70 kg, and may be administered once a day It may be administered in divided doses.

 Health functional food and health food composition for prevention or improvement of liver disease

The present invention provides a health functional food and a health food composition comprising Gomisin G and / or Gomisin O as an active ingredient.

There are no particular restrictions on the type of food, but examples of foods to which the gomisin G and / or gomisin O of the present invention can be added include drinks, meat, sausage, bread, biscuits, rice cakes, There are dairy products including chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, soups, beverages, alcoholic drinks and vitamin complexes, dairy products and dairy products. And health functional foods.

The health food and health functional food composition containing Gomisin G and / or Gomisin O according to the present invention can be added directly to food or can be used together with other food or food ingredients, May be appropriately used depending on the method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). Generally, the amount of gomisin G and / or gomisin O in the health food and the health functional food may be 0.1 to 90 parts by weight of the total food weight. However, in the case of long-term intake for the purpose of health maintenance or health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The health food and health functional food composition of the present invention is not particularly limited to the other ingredients other than the present invention Gomisin G and / or Gomisin O as essential ingredients in the indicated ratios, Various flavoring agents, natural carbohydrates, and the like as additional components. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above . The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 health functional food composition of the present invention.

In addition to the above, the health food and health functional food composition containing Gomisin G and / or Gomisin O according to the present invention may be used in various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors (Such as cheese and chocolate), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonated drinks A carbonating agent to be used, and the like. In addition, the health food and health functional food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.

These components may be used independently or in combination. The proportion of such additives is not critical, but is in the range of from 0.1 to about 20 parts by weight per 100 parts by weight of the health food and health functional food composition containing Gomisin G and / or Gomisin O of the present invention . ≪ / RTI >

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

<Preparation Example 1> Mouse animal model inducing liver damage using TAA

As shown in FIG. 1, mice were injected with phosphate-buffered saline (PBS) buffer solution for 10 days and then liver was harvested 8 weeks later. In the TAA treated group, 10 days after administration of PBS buffer, 100 mg / kg of TAA was intraperitoneally injected three times per week for 8 weeks to induce liver injury. In the Omiza + TAA treatment group, Oriza extract was orally administered at a dose of 300 mg / kg. After 10 days, the Omiza extract was administered orally and the TAA was intraperitoneally administered for 3 weeks for 8 weeks. After 8 weeks of TAA treatment, liver tissue was removed after blood collection.

&Lt; Example 1 > Preparation of Omija extract

3 kg of ethanol was added to 1 kg of dried omelet in the shade, and then immersed for 3 days. After extracting only liquid components with filter paper, it was dried with a vacuum concentrator and again lyophilized to prepare an extract of Omija.

Example 2: Preparation of fractions of Omiza extract

Fractions according to the polarity of the ethanol extract of Example 1 were sequentially fractionated using hexane, chloroform, ethyl acetate and butanol to obtain five kinds of fraction layers including an aqueous solution layer. The ethanol extracts were dried with a vacuum concentrator and then lyophilized to prepare fractions.

&Lt; Experimental Example 1 >

Analytical equipment was analyzed by high performance liquid chromatography (HPLC) Agilent 1260 Infinity, Symmetry 4.6 X 250mm 5um as column and 220nm UV / VIS detector. The mobile phase used 65% acetonitrile. The hexane layer fractions were used for HPLC analysis. 9 authentic gomisin derivatives were used. The nine gomisin derivatives were Gomisin D, Gomisin G, Gomisin H, Gomisin J, Gomisin N, Gomisin O, Angeloylgomisin H, Angeloylgomisin O, and Benzoylgomisin O (see FIG.

The hexane fraction of Omija was analyzed under the same conditions as the authentic sample. Gomisin G, Gomisin J, Gomisin N, Gomisin O, Angeloylgomisin H, and Angeloylgomisin O (see FIG. 9).

EXPERIMENTAL EXAMPLE 2 Protective Effect of Schizandra chinensis Extract on Hepatic Function in Mice Induced by Liver Damage by Thioacetamide (TAA)

Thioacetamide (TAA) is a chemical that induces chronic hepatic damage and causes liver fibrosis.

The enzyme alanine aminotransferase (ALT), also known as glutamic pyruvate transaminase (GPT), and the aspartate aminotransferase (AST), aka glutamic oxaloacetic transaminase (GOT) When hepatocytes are damaged, they are released into the blood and the blood concentration increases. Therefore, changes in serum ALT and AST enzyme activity are used as indirect indicators of liver function impairment.

To investigate the effect of Omija extract on inhibition of hepatic function by TAA, the experiment was divided into three groups as shown in Fig. 1, and the changes of ALT and AST enzyme activity were measured in mouse serum.

The ALT and AST enzyme activities were purchased from Shenzhen Mindray Bio-Medical Electronics Company (Shenzhen, P.R. China) and used as recommended in the instruction manual of the kit. 1 mL of ALT and AST substrate solution and 0.1 mL of mouse serum sample were added to the test tube and the absorbance at 340 nm was measured.

As a result, ALT enzyme activity was 57.83 + 13.93, 665 + 390.4, 292.8 + 192.7 Unit / L in the control group, TAA treatment group and Omija extract + TAA treatment group And 55.67 + 11.67, 158 + 57, and 73.5 + 19.34 Unit / L, respectively (Table 2). The efficacy of Omija extract inhibiting ALT and AST enzyme activity increased by TAA was statistically significant (P <0.05, statistical significance was tested by the Sidak's multiple comparison test after ANOVA). These results confirm that the extract of Omija has the effect of protecting liver function damaged by TAA.

Experimental Example 3: Inhibition of hepatic fibrosis of Schizandra chinensis extract in mice induced by hepatic injury with thioacetamide (TAA)

As shown in Fig. 1, liver tissues extracted from the control group, TAA alone treatment group and TAA + Omija extract treatment group were fixed with 10% neutral formalin and paraffin blocks were prepared in paraffin. The paraffin tissue was cut into 4 ㎛ sections. The paraffin tissues were attached to the slides and the paraffin removal and hydration steps were performed stepwise and the liver tissues were stained with hematoxylin and eosin staining (H & E staining). After the paraffin removal and hydration process, staining of collagen fibers in tissues was stained with Bouing Fluid, Heamatoxylin, Blebrich Xcarlet / Acid Fushsin solution, Phosphomolybdic / Phosphotungstic Acid Solution, Aniline Blue Solution and Acetic Acid Solution. Collagen fiber staining was again verified using Sirius Red Staining.

As a result, hepatocyte damage was significantly observed in the TAA treated group compared to the control group. In the experimental group administered with the extract of OMIZA, the liver tissue damage by TAA treatment was markedly reduced (see the upper part of FIG. 3). Collagen fiber deposition in the control group was scarcely found, but in TAA-treated liver tissue, collagen fibers were predominantly centered around the central vein veins (see second line on the right side of FIG. 3). In the experimental group administered with the extract of the omija, it was observed that the collagen fibers accumulated by the TAA treatment were remarkably reduced (see the right third line of Fig. 3).

Therefore, it was confirmed that the extract of Omija inhibited the deposition of collagen fibers induced by liver injury and inhibited the process of liver fibrosis.

<Experimental Example 4> Inhibitory effect of Schizandra chinensis extract on collagen gene expression in mice induced by hepatic injury with thioacetamide (TAA)

The key response in hepatic fibrosis is the early stage of promoting collagen fiber synthesis by activating hepatic stellate cells (HSC) by cytokines such as TGF-β secreted from damaged hepatocytes or Cooper cells. When HSCs are stimulated, the expression of α-smooth muscle actin (α-SMA) is increased by differentiation into myofibroblast-like cells (Front Biosci, 7 (2002) pp. d793-d807). In the present invention, the gene expression of the first type A1 collagen fiber (COL1A1) expressed in hepatic stellate cells (HSC) is evaluated as a molecular index for measuring the activity of hepatic stellate cells (HSC) Respectively.

As shown in FIG. 1, 20 mM HEPES (pH 7.2), 1% Triton X-100, 10% glycerol, 150 mM NaCl, and 10 μg / ml leupeptin were added to the liver tissues extracted from the control group, TAA alone treatment group and TAA + Cell lysis buffer containing 1 mM PMSF was added and pulverized using a homogenizer (Kinematica AG, Polytron PT2500E). The supernatant was harvested by centrifugation at 12,000 rpm for 30 minutes in a tabletop centrifuge, and a sample prepared to contain the same amount of protein was subjected to SDS-polyacrylamide gel electrophoresis to remove the proteins present in the cells Respectively. The proteins separated by electrophoresis were transferred to a polystyrene membrane, reacted for 5 hours with a primary antibody that specifically recognizes the target protein, and then incubated with a mustard radish peroxidase-conjugated secondary antibody capable of recognizing the primary antibody Horseradish peroxidase-conjugated secondary antibody was reacted for 1 hour, and then the product was exposed to X-ray film using a chemiluminescence detection system (Amersham Pharmacia Biotech, Piscataway, NJ, USA) The amount of protein that was changed was analyzed. Primary antibodies recognizing α-SMA and type 1 A1 collagen fibers (COL1A1) were purchased from Dakocytomation and Boster, respectively. Secondary antibodies were purchased from Cell Signaling Technology.

As a result, as shown in Fig. 4, in the liver tissue of the TAA alone-treated mice, the amount of protein of the α-SMA and the type 1 A1 collagen fiber (COL1A1) was increased, but in the experimental group administered with TAA, The amount of protein expression was significantly reduced in comparison with the TAA-treated group. However, the amount of protein in the housekeeping gene, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), did not change. These results indicate that the extract of Omija inhibits the differentiation of hepatic stellate cells (HSC) and reduces the protein accumulation of type I A1 collagen fiber (COL1A1).

<Experimental Example 5> Inhibitory effect of α-SMA and collagen gene mRNA gene expression in Schizandra chinensis extract (HSC) using reverse transcription polymerase chain reaction

We investigated whether the inhibitory effect of α-SMA and type I A1 collagen fiber (COL1A1) on the protein expression of hepatic stellate cells (HSC) in liver tissues damaged by TAA was observed. When liver tissue is damaged, TGFβ is secreted from hepatocytes or Cooper cells, activating hepatic stellate cells (HSC) and promoting hepatic fibrosis (Proc Natl Acad Sci U S, 96 (1999), pp. 2345-2349).

The expression of α-SMA and type-I A1 collagen fiber (COL1A1) mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) using 10 μg / mL TGFβ in HSC. RT-PCR).

Hepatic stellate cells (HSCs) were purchased from the American Type Culture Collection (ATCC) and cultured in DMEM supplemented with 10% fetal bovine serum (Invitrogen Life Technologies), antibiotic (Antibiotic-Antimycotic solution, purchased from Invitrogen Life Technologies) (Invitrogen Life Technologies) culture medium at 37 ° C in a 5% CO ₂ incubator at a seeding density of 1 × 10 ⁶ in a 100-mm cell culture dish once every two days. Total RNA isolation was performed using TRIzol RNA Isolation Reagents (purchased from QIAGEN). For the double-stranded cDNA synthesis for reverse transcription, 0.5 μg of total RNA was synthesized using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) and synthesized according to the manufacturer's recommendation method and 0.0125 μg of double- PCR was performed. Primer base sequences for gene amplification were synthesized on the basis of already known gene base sequences in Macrogen Company (Seoul, Korea) as shown in Table 1 below. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was amplified as a control gene to compare the relative amounts of each experimental group. The PCR reaction was 94, 1 min after DNA denaturation at 95, 5 min. 65, 2 min; And 70, for 1 minute were repeated 30 times. The PCR products were electrophoresed on 1% agarose gel and stained with EtBr (ethidium bromide).

As a result, stimulation of TGFβ with 10 ng / mL of hepatic stellate cells (HSC) as shown in FIG. 5A resulted in an increase in the expression of α-SMA and type 1 A1 collagen gene (COL1A1) gene after 6 hours . Cells were harvested at 12 and 10 μg / mL for 30 min, treated with TGFβ at a concentration of 10 ng / mL, and subjected to reverse transcription-polymerase chain reaction (RT-PCR) ). As a result, as shown in FIG. 5B, the extract of Omiza decreased the expression of α-SMA and type 1 A1 collagen gene (COL1A1) mRNA increased by TGFβ in a concentration-dependent manner.

The primer base sequence used in the reverse transcription polymerase chain reaction gene Standing column α-SMA forward 5'-CTGAGCGTGGCTATTCCTT-3 '(SEQ ID NO: 1) reverse 5'-CTTCTGCATCCTGTCAGCAA-3 '(SEQ ID NO: 2) COL1 forward 5'-GACGCCATCAAGGTCTACTG-3 (SEQ ID NO: 3) reverse 5'-ACGGGAATCCATCGGTCA-3 '(SEQ ID NO: 4) Actin forward 5'-TGGAATCCTGTGGCATCCATGAAAC-3 '(SEQ ID NO: 5) reverse 5'-TAAAACGCAGCTCAGTAACAGTCCG-3 '(SEQ ID NO: 6)

Experimental Example 6 Inhibitory Effect of Omiza Extract on α-SMA and Collagen Gene mRNA Expression by Quantitative Real-time Polymerase Chain Reaction (QRT-PCR) in Hepatic Stem Cells (HSC)

Quantitative real-time polymerase chain reaction (QRT-PCR) is a quantitative method based on polymerase chain reaction. That is, it can measure the amount of mRNA accurately by simultaneously measuring the amplification degree and the expression amount of the target gene. CDNA was synthesized as described above, and then a quantitative real-time PCR experiment was performed by purchasing a TaqMan-iQ supermix kit (Bio-Rad). 5'-6-FAM-CATCGTGGCTTCTCTGGTCTCC -BHQ-1-3 'was used as the TaqMan probe sequence for measuring the mRNA of the type 1 A1 collagen (COL1A1) gene and the TaqMan probe sequence used for the measurement of the GAPDH mRNA as a control gene Was 5'-Yakima Yellow-CGTCGCCAGCCGAGCCACATCGC-BHQ-1-3 '(SEQ ID NO: 8). Cells were harvested at 0, 6, 12, and 24 hours after stimulation with hepatic stellate cells (HSC) 10 ng / mL of TGFβ and quantitative real-time PCR was performed.

The expression of type 1 A1 collagen (COL1A1) gene mRNA was maximal at 6 hours after TGFβ treatment, 4.3 times higher than that of the control without any treatment. Thereafter, the expression amount of the type 1 A1 collagen (COL1A1) gene mRNA gradually decreased in a time-dependent manner (see FIG. 6A). 10 and 20 μg / mL of Omija extract were pretreated for 30 minutes, treated with 10 ng / mL of TGFβ, and harvested after 6 hours to perform quantitative real-time PCR. As a result, the Omija extract at the concentration of 10 and 20 μg / mL decreased the expression of the type 1 A1 collagen gene (COL1A1) mRNA, which was increased 3.8-fold by TGFβ compared with the control, to 2-fold and 1.5-fold, respectively ).

Thus, it has been confirmed that the extract of Omija reduces the expression of type I A1 collagen gene (COL1A1) mRNA, which is expressed by TGFβ stimulation in hepatic stellate cells (HSC).

<Experimental Example 7> Fractional analysis of Omiza extract showing inhibitory effect on fibrosis

 In order to analyze the inhibitory effect of each fraction layer on hepatic fibrosis, it was treated with hepatic stellate cells (HSC) as in the above Experimental Example, and the inhibitory effect of α-SMA and type 1 A1 collagen (COL1A1) gene mRNA expression increased by TGFβ Respectively.

As a result, it was confirmed that the ethanol, hexane, ethyl acetate, and butanol fraction layers inhibited the expression of α-SMA and type 1 A1 collagen (COL1A1) gene mRNA expressed by TGFβ as shown in FIG. Thereafter, a hexane fraction layer was used to separate the active components.

<Experimental Example 8> Separation of effective components showing the effect of inhibiting hepatic fibrosis in the fraction of Omija hexane

Gomisin G, Gomisin N, Gomisin O, Angeloylgomisin H, and Angeloylgomisin O isolated from the fraction of Omija hexane inhibited the hypersensitivity effect of α-SMA and the type 1 A1 collagen gene (COL1A1) gene . Gomisin D, Angeloylgomisin H and Butanoloyolgomisin O were used as control.

Various HSCs were treated with hepatic stellate cells (HSCs) to examine the inhibitory effect of α-SMA and type 1 A1 collagen (COL1A1) gene mRNA expressed by TGFβ. As a result, as shown in Fig. 10, all of the six hypermuscine derivatives present in the omizahexane layer inhibited the expression of mRNA of? -SMA and type 1 A1 collagen (COL1A1) genes, which are increased by TGF ?.

Meanwhile, the hypericin G and / or hypericin O according to the present invention can be formulated into various forms according to the purpose. Hereinafter, the inventors of the present invention have exemplified several formulations containing the hypermystin G and / or hypericin O as an active ingredient, and the present invention is not limited thereto.

&Lt; Formulation Example 1 > Preparation of powders

Hypermishin G and / or hypericin O 2 g

Lactose 1 g

The above components were mixed and packed in airtight bags to prepare powders.

&Lt; Formulation Example 2 > Preparation of tablet

Hypermishin G and / or hypericin O 100 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

&Lt; Formulation Example 3 > Preparation of capsules

Hypermishin G and / or hypericin O 100 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

&Lt; Formulation Example 4 > Preparation of injection

Hypermishin G and / or hypericin O 100 mg

Mannitol 180 mg

Na ₂ HPO ₄ .2H ₂ O 26 mg

Distilled water 2974 mg

According to the conventional method for preparing an injectable preparation, an injectable preparation was prepared by incorporating the aforementioned components in the amounts indicated.

Manufacturing Example of Health Food

The hyperinsin G and / or hyperinsin O according to the present invention can be manufactured into various types of health food according to the purpose. The following are illustrative examples of a method for producing some health foods containing hyperinsin G and / or hyperinsin O according to the present invention as an active ingredient, and the present invention is not limited thereto.

<Health food production example 1> Manufacture of dairy products

0.01 to 1 part by weight of the hyperinsin G and / or hyperinsin O of the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

&Lt; Health food production example 2 >

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh. Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer. The hyperinsin G and / or hyperinsin O of the present invention was concentrated under reduced pressure in a vacuum concentrator to obtain a dry powder. The above-prepared grains, seeds, and dried powders of hypericin G and / or hypericin O were blended in the following proportions.

Grain (34 parts by weight of brown rice, 19 parts by weight of yulmu, 20 parts by weight of barley)

Seeds (7 parts by weight of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds)

(2 parts by weight) of hypericin G and / or hypericin O,

(1.5 parts by weight), and

Rhizome (1.5 parts by weight).

Manufacturing Example of Health Functional Food

The hypericin G and / or hypericin O according to the present invention can be manufactured into various types of health functional foods according to the purpose. The following are illustrative examples of the method for preparing some health functional foods containing the hyperinsin G and / or hyperinsin O according to the present invention as an active ingredient, and the present invention is not limited thereto.

&Lt; Health functional food production example 1 > Production of health functional food

Hypermishin G and / or hypericin O 100 mg

Vitamin mixture Suitable amount

Vitamin A acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 μg

Vitamin C 10 mg

Biotin 10 μg

Nicotinic acid amide 1.7 mg

Folic acid 50 μg

Calcium pantothenate 0.5 mg

Mineral mixture Suitable amount

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dicalcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a component suitable for a health functional food as a preferred embodiment, the compounding ratio may be arbitrarily changed, and the above components may be mixed , Granules may be prepared and used in the manufacture of health functional food compositions according to conventional methods.

&Lt; Health Functional Food Production Example 2 >

Hypermishin G and / or hypericin O 100 mg

Citric acid 100 mg

oligosaccharide 100 mg

Plum concentrate 2 mg

Taurine 100 mg

Purified water is added to the entire 500 mL

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the solution thus prepared was filtered to obtain a sterilized container. The resulting solution was sealed and sterilized, &Lt; / RTI &gt; Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (12)

A pharmaceutical composition for preventing or treating liver disease comprising at least one of Gomisin G and Gomisin O. The method according to claim 1,
Wherein the liver disease is liver cirrhosis, liver fibrosis, acute hepatitis, chronic hepatitis or liver cancer. 3. The method of claim 2,
Wherein the liver disease is liver fibrosis. The method according to claim 1,
Wherein said composition inhibits the expression of type I collagen gene (COL1A1) and alpha-smooth muscle actin (alpha-SMA, alpha-smooth muscle actin) in hepatic stellate cells. A health functional food composition for preventing or ameliorating liver disease comprising at least one of Gomisin G and Gomisin O. 6. The method of claim 5,
Wherein the liver disease is liver cirrhosis, liver fibrosis, acute hepatitis, chronic hepatitis or liver cancer. The method according to claim 6,
Wherein the liver disease is liver fibrosis. 6. The method of claim 5,
Wherein said composition inhibits the expression of type I collagen gene (COL1A1) and alpha-smooth muscle actin (alpha-SMA, alpha-smooth muscle actin) in hepatic stellate cells. A health food composition for preventing or ameliorating a liver disease comprising at least one of Gomisin G and Gomisin O. 10. The method of claim 9,
Wherein the liver disease is liver cirrhosis, liver fibrosis, acute hepatitis, chronic hepatitis or liver cancer. 11. The method of claim 10,
Wherein the liver disease is liver fibrosis. 10. The method of claim 9,
Wherein said composition inhibits the expression of type I collagen gene (COL1A1) and alpha-smooth muscle actin (alpha-SMA, alpha-smooth muscle actin) in hepatic stellate cells.

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