WO2014193088A1 - Composition for preventing or treating hepatitis c including vitidis vinferae radix extract or fraction thereof as active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing or treating hepatitis C including, as an active ingredient, a Vitidis Vinferae Radix extract or a fraction thereof and, more specifically, to a Vitidis Vinferae Radix extract which is extracted with water and alcohol or a mixed solvent thereof, or a fraction thereof, which have remarkably low hepatotoxicity and exhibit an excellent effect of selectively inhibiting genome replication of a hepatitis C virus, and thus can be useful for preventing or treating hepatitis C.

Description

Composition for the prevention or treatment of hepatitis C comprising grape root extract or fractions thereof as an active ingredient

The present invention relates to a composition for the prevention or treatment of hepatitis C, comprising grape root extract or fractions thereof as an active ingredient.

Liver cancer and liver disease are one of the four most common deaths per 100,000 Korean population, along with cerebrovascular disease, heart disease and respiratory disease. Hepatitis B and C are caused by viral hepatitis B viruses. This accounts for 83% of the total. Among them, hepatitis C virus is a medically important pathogen with about 170 million infected people worldwide. Hepatitis C was classified as non-A non-B post-transfusion associated hepatitis (NANB) until the mid-1980s. It has been found that the disease caused by, and since then, active research and development has been progressed.

Hepatitis C infection by the hepatitis C virus is fatal in most cases because it progresses to chronic hepatitis and develops into chronic liver diseases such as cirrhosis and liver cancer over a long period of 15 to 20 years, and hepatitis C virus (HCV). , Hepatitis C virus) has been reported to cause about 8,000 to 10,000 deaths each year in the United States alone. In particular, most of the terminal hepatitis C patients die from waiting for liver transplantation and do not receive a liver transplant. Statistically, 1.5% of the population in Korea are infected with hepatitis C virus.

Hepatitis C Virus (HCV) is the only virus classified among the hepaciviruses in the flaviviridae family. Hepatitis C Virus (HCV) has a single strand of RNA consisting of approximately 9600 nucleic acids as its viral genome. have. The RNA is translated into a polyprotein consisting of about 3000 amino acids in hepatocytes, and the translated polyprotein is a non-structural protein of a signal peptidase and virus present in the endoplasmic reticulum of the hepatocytes. It is expressed by 10 different viral proteins by one NS3 protease. Among the expressed viral proteins, structural proteins such as envelope glycoproteins E1, E2 and capsid proteins core are used to make particles of the virus, NS2, Non-structural proteins such as NS3, NS4A, NS4B, NS5A, and NS5B are used to create the viral genome replication complex required to replicate the virus's genome. HCV replicates the viral RNA genome by creating a viral genome replication complex in the membrane that occurs in the endoplasmic reticulum. Viral genomic replication complexes retain their invaginated accumulation in the membrane, creating independent structures isolated from the outside environment, expressing the nonstructural proteins of the virus, and expressing the nonstructural proteins expressed in the RNA RNA polymerase. Together with the NS5B protein, it acts as a genome replicator of the virus. However, it is not known how the virus genome replication complex is made and maintained. Among the nonstructural proteins of the virus, in particular the NS4B protein has been found to play the most critical role in making the membranous web, a multi-vesicular structure necessary for the genome replication of the virus in hepatocytes. It is believed that the structure of the membrane web made by NS4B provides the physical structure necessary to make the viral genome replication complex. In addition, the NS5A protein assembles the viral genome replication complex using non-structural proteins of other viruses, transfers the resulting viral genome into a structure called lipid droplets, where it binds the viral core protein with the viral genome, It is known to play an important role in making viral particles. In particular, protein-protein interactions between viral non-structural proteins are known to play a decisive role in the construction of viral genome replication complexes, but the exact role of them at the molecular level is still unknown.

Types of antiviral agents that can treat viral diseases include indirect-acting antivirals (IAAs) that attack viruses indirectly, depending on the mechanism of action of the antiviral agents, and direct-acting antivirals that attack viruses directly. , DAAs). Interferon alpha, which is secreted by the body when a pathogenic external microorganism such as a virus penetrates the body, is a representative antiviral intrinsic immunity enhancer that indirectly attacks the virus, rather than attacking the virus itself. Because it blocks the proliferation of virus cells by increasing the effect, it is effective for various kinds of viral diseases and shows antiviral activity by controlling the function of protein of host cell in which virus is parasitic. The probability of developing a mutant virus is relatively low. However, to overcome this, antiviral specificity, which can only show antiviral activity against a particular virus, is significantly reduced, so in order to overcome this, a much larger amount of interferon than the amount of interferon naturally secreted in vivo is used for therapeutic purposes. Administration, thus causing serious side effects and toxicity such as suicidal thoughts, depression, anemia and the like.

On the other hand, anti-viral agents that directly attack the virus, unlike interferon, selectively inhibits only the specific functions of the viral proteins essential for life history of the virus, thereby exhibiting high anti-viral activity even at relatively low concentrations. In addition, compared to antiviral agents that indirectly attack the virus, it is characterized by low toxicity and high safety. Reverse transcriptase inhibitors and protease inhibitors of human immunodeficiency virus (HIV) are representative antiviral agents that directly attack the virus. It inhibits the proliferation of virus by inhibiting enzymes and proteolytic enzymes. However, there is a relatively high possibility that the virus may show resistance and resistance to existing antiviral agents by changing the amino acid sequence of the viral protein targeted by the antiviral agent, especially RNA polymerase of hepatitis C virus. Since there is no 5'-3 'endonuclease activity that corrects nucleic acids that are misplaced during replication of the RNA genome, the mutant virus is easily generated during the replication of the hepatitis C virus genome, RNA. Resistant and resistant to antiviral agents that directly attack

Interferon-alpha and ribavirin, which are currently commonly used for the treatment of hepatitis C, are indirect-acting antivirals (IAAs) that indirectly attack viruses by enhancing innate immunity and inhibiting nucleic acid biosynthesis. Even when the two drugs are used together, the sustained virologic response (SVR) after 52 weeks of intravenous injection and oral administration is less than 50% depending on the genotype of the virus. In addition, many hepatitis C patients give up treatment due to serious side effects and toxicity such as suicidal thoughts, depression and anemia. Therefore, there is an urgent need for a more effective and safe drug development that can replace the antiviral drugs for the existing hepatitis C virus.

On the other hand, Vitidis Vinferae Radix is a herb that is used to dry the roots of grapes (Vitis vinifera) and wild grapes (Vinus amurensis) belonging to the family Vitaceae in the sun or fresh. Radix (Vitidis Vinferae Radix). In Dongbogam, “If you put it on the water and drink it, you'll feel sick and sick. And after pregnancy, Tajigi dries down when it dries the teeth. ”It has been recorded since ancient times, and it has been used for the treatment of rheumatism, pain, swelling, and urinary bleeding.

As described above, various pharmacological effects of grape roots are known, but the prevention, improvement or treatment effect of hepatitis C of grape roots is not known, and there is no research on this.

Accordingly, the present inventors have continued efforts to develop novel hepatitis C therapeutics. As a result, the grape root extract or its fractions are excellent in selectively inhibiting the genome replication of the hepatitis C virus, while having very low toxicity to hepatocytes. By confirming the effect, the present invention was completed.

An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of hepatitis C, including grape root extract or fractions thereof as an active ingredient.

Still another object of the present invention is to provide a food composition for preventing or improving hepatitis C, which comprises grape root extract or a fraction thereof as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of hepatitis C or a food composition for the prevention or improvement of hepatitis C, comprising the extract of Vitidis Vinferae Radix or fractions thereof as an active ingredient. to provide.

In one embodiment of the present invention, the grape root extract is characterized in that the grape root is extracted with water, alcohol having 1 to 4 carbon atoms or a mixed solvent thereof.

In another embodiment of the present invention, the C1-4 alcohol is characterized in that methanol or ethanol.

In another embodiment of the present invention, the fraction is characterized in that the ethyl acetate fraction, butanol fraction, hexane fraction or water fraction obtained by sequentially fractionating the grape root extract with ethyl acetate, butanol, hexane, water.

In another embodiment of the present invention, the grape root extract or fraction is characterized in that it has a selective inhibitory activity of RNA genome replication of hepatitis C virus.

The grape root extract or fractions thereof according to the present invention have a very low toxicity to hepatocytes, but have an excellent effect of selectively inhibiting the genome replication of hepatitis C virus, which is useful for the prevention or treatment of hepatitis C. Can be.

1 is a diagram showing the effect of the grape root methanol extract of the present invention on RNA genome replication and cell viability of hepatitis C virus (●: cell survival rate, △: HCV genome replication rate).

2 is a diagram illustrating the genomic structure of the HCV replicon FL-J6 / JFH-5C19Rluc2AUbi HCV reporter virus used to measure the genomic replication of hepatitis C virus and the principle of viral genome replication inhibitor discovery.

3 is a diagram showing the effect of the grape root ethyl acetate fraction of the present invention on RNA genome replication and cell viability of hepatitis C virus (●: cell survival rate, ■: HCV genome replication rate).

4 is a diagram showing the effect of the grape root butanol fraction of the present invention on RNA genome replication and cell viability of hepatitis C virus (●: cell survival rate, ■: HCV genome replication rate).

5 is a diagram showing the effect of the grape root ethanol extract of the present invention on RNA genome replication and cell viability of hepatitis C virus (●: cell survival rate, ■: HCV genome replication rate).

6 is a diagram showing the effect of the grape root hexane fraction of the present invention on the RNA genome replication and cell viability of hepatitis C virus (●: cell survival rate, ■: HCV genome replication rate).

7 is a diagram showing the effect of the grape root water extract of the present invention on RNA genome replication and cell viability of hepatitis C virus (●: cell survival rate, ■: HCV genome replication rate).

8 shows the genome structure of the subgenomic replicon Bart79I-NS5A-GFP HCV reporter virus used to measure protein expression of hepatitis C virus.

FIG. 9 is a graph illustrating the effect of the grape root methanol extract of the present invention on the protein expression of hepatitis C virus in GFP-expressed hepatocytes containing tagging subgenomic replicon Bart79I-NS5A-GFP. The amount of fluorescence was measured using a FACS (fluorsecence assorted cell sorter).

Figure 10 is the effect of the grape root methanol extract of the present invention on protein expression of hepatitis C virus was measured by Western blot the amount of viral NS5A protein and host cell beta actin protein.

The present invention provides a composition for the prevention or treatment of hepatitis C, comprising grape root (Vitidis Vinferae Radix) extract or a fraction thereof as an active ingredient.

The composition comprises a pharmaceutical composition or a food composition.

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

Grape root extract as an active ingredient in the composition of the present invention can be obtained by the following method.

First, it is washed thoroughly with water, dried and then ground. The grape root may be grown or commercially available, without limitation. 1 to 10 times, preferably 2 to 5 times the volume of the solvent is added to the pulverized grape root to be completely immersed, and then extracted for 1 to 5 hours, preferably about 2 hours. The extraction solvent is not limited thereto, but one or more solvents selected from water, alcohols having 1 to 4 carbon atoms, and mixed solvents thereof may be used, and preferably methanol or ethanol. The extraction method may be an ultrasonic extraction method, a subcritical extraction method, a high temperature extraction method, a high pressure extraction method, a filtration method or a reflux extraction method, etc., but a conventional extraction method in the art may be used, but it is preferable to use an ultrasonic extraction method, but is not limited thereto. . Thereafter, the grape root extract was separated by centrifugation, the supernatant was collected and filtered, and the filtrate was concentrated under reduced pressure, and then freeze-dried to obtain a powdered grape root extract. In addition, the grape root extract is suspended in distilled water, and then sequentially fractionated using ethyl acetate, butanol and hexane to obtain the remaining distilled water solubles.

Grape root extract according to the present invention has a very low toxicity to hepatocytes, but has an excellent effect of selectively inhibiting the genome replication of hepatitis C virus, it can be usefully used for the prevention or treatment of hepatitis C.

The composition of the present invention may further contain at least one known active ingredient having a prophylactic or therapeutic effect of hepatitis C with grape root extract or a fraction thereof.

The composition of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. It may also be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like in the form of conventional formulations, external preparations, suppositories, and sterile injectable solutions. Suitable formulations known in the art are preferably those disclosed in Remington's Pharmaceutical Science, recently, Mack Publishing Company, Easton PA.

Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose , Microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. In formulating the composition, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, lactose, It is prepared by mixing gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be used. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like may be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

As used herein, the term "administration" means providing a subject with any of the compositions of the present invention in any suitable manner.

Preferred dosages of the pharmaceutical compositions of the present invention depend on the condition and weight of the individual, the extent of the disease, the form of the drug, the route of administration and the duration and can be appropriately selected by those skilled in the art. For the desired effect, the grape root extract or fractions thereof of the present invention may be administered in an amount of 0.1 mg / kg to 1000 mg / kg per day, preferably in an amount of 0.001 to 200 mg / kg, once or several times a day. It may be administered in divided doses.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections.

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

In the present invention, the health functional food refers to a food having a bioregulatory function, such as prevention or improvement of disease, biological defense, immunity, recovery of the disease, inhibition of aging, and should be harmless to the human body when taken in the long term. Grape root extract of the present invention may be added to dietary supplements for the purpose of preventing or improving hepatitis C. When the grape root extract of the present invention is used as a food additive, the grape root extract may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, the grape root extract of the present invention in the preparation of food or beverage is added in an amount of up to 15% by weight, preferably up to 10% by weight relative to the raw material. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, it may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

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

The health beverage composition of the present invention may include various flavors or natural carbohydrates, and the like as an additional ingredient, as in a general beverage. The natural carbohydrates described above may be used as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and natural sweeteners such as dextrin and cyclodextrin, and synthetic sweeteners such as saccharin and aspartame. The proportion of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, And carbonating agents used in the carbonated beverage. In addition, the composition of the present invention may include a pulp for the production of natural fruit juice, fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not critical but is usually selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

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

EXAMPLE

Example 1 Preparation of Grape Root Water Extract

Grape root (Gyeongdong Market, Deokhyeondang) was washed with water, dried and ground. 500 ml of water was added to 100 g of the pulverized grape root, soaked for 2 hours, and extracted by ultrasonic extraction. Thereafter, the obtained grape root extract was centrifuged to separate the supernatant, collected and filtered, and the filtrate was concentrated under reduced pressure and lyophilized to obtain a powdered grape root extract (14.1 g).

Example 2. Preparation of Grape Root Methanol Extract

A grape root methanol extract (9.1 g) was obtained in the same manner as in Example 1, except that 100% methanol was used instead of water in Example 1.

Example 3. Preparation of Grape Root Fractions

Except for using ethanol instead of water in Example 1, grape root ethanol extract was obtained in the same manner as in Example 1 (8.3g), the obtained grape root ethanol extract was suspended in distilled water, hexane, ethyl acetate, Fractions were sequentially performed with butanol and water to obtain a hexane fraction (0.7 g), an ethyl acetate fraction (2.5 g), a butanol fraction (2.1 g) and a water fraction (3 g).

Experimental Example 1. Analysis of liver cytotoxicity of grape root extract

In order to confirm the liver cytotoxicity of the grape root methanol extract obtained in Example 2, the following experiment using EZ-Cytox cell viability assay (EZ-Cytox cell viability assay, Daeil Lab Service) Was performed.

First, 1.7 × 10 ⁴ to 2.0 × 10 ⁴ Huh 7.5 human liver cancer cells per well were added to a 96 well-plate and incubated for 24 hours in a 37 ° C. incubator. The cells were treated with grape extract methanol extract of various concentrations (0.1 ng / ml ~ 10 μg / ml) and then incubated for 72 hours. As a control, DMSO (dimethyl sulfoxide) was treated. Subsequently, discard the culture medium, wash with PBS, and add 100 μl of 1/10 (v / v) dilution prepared by adding the cell culture solution to the EZ-Cytox reagent containing water-soluble tetrazolium salt. The reaction was carried out for a time. After the reaction was completed, the absorbance of the cells was measured at a wavelength of 450 nm using a spectrophotometer. Based on the absorbance at the time of DMSO treatment to 100, relative absorbance at the treatment of grape root methanol extract was calculated, the results are shown in FIG.

As a result, as shown in Figure 1, the grape root extract according to the present invention showed a very good cell viability in the range of 0.1 ng / ml to 10 μg / ml, there is little toxicity to liver cells and high safety Confirmed.

Experimental Example 2. Measurement of Hepatitis C Virus Genome Replication Inhibitory Activity of Grape Root Extract or Fraction

To determine the hepatitis C virus genome replication inhibitory activity of the grape root methanol extract obtained in Example 2 or the grape root ethanol extract obtained in Example 3 and fractions thereof, the following experiment was performed.

2-1. Preparation of Reporter Virus to Measure Genome Replication of Hepatitis C Virus

In order to measure genome replication of hepatitis C virus, FL-J6 / JFH-5C19Rluc2AUbi HCV replicon belonging to genotype 2a was used as a reporter virus. The genomic structure diagram of the reporter virus is shown in FIG.

As shown in FIG. 2, the reporter virus contains the entire genome of hepatitis C virus belonging to genotype 2a in a cDNA state, and has an internal ribosome entry site (IRES) and a core of the virus. ) Between the protein is attached a Ubi sequence of Lenilla luciferase and a self-cleaving foot and mouth disease virus 2A protein, thereby providing the FL-J6 / JFH-5C19Rluc2AUbi plasmid. When HCV RNA obtained by in vitro transcription using T7 RNA polymerase was transfected into Huh7.5 cells, a liver cancer cell line, the RNA of the injected virus enters an internal ribosome. Polyproteins are produced through translation using position (IRES). Renilla luciferase in the produced multiprotein is separated from viral non-structural proteins with the help of the Ubi sequence of the self-cutting foot disease virus 2A protein, and indirectly in the hepatocytes by measuring the activity of the isolated lenilla luciferase. Viral RNA genome replication can be measured.

2-2. Determination of Hepatitis C Virus Genome Replication Inhibitory Activity of Grape Root Extract or Fraction

Huh7.5 human liver cancer cells were trypsinized and resuspended with a PBS solution to a cell density of 1.5 × 10 ⁷ / ml. A total of 5 μg of in vitro transcribed FLJ6 / JFH-5 C19Rluc2AUbi RNA was mixed with 400 μl of PBS buffer containing Huh 7.5 human liver cancer cells and placed in 2-mm-gap cuvette (BTX). The FL-J6 / JFH-5 C19Rluc2AUbi RNA was transfected into Huh7.5 human liver cancer cells by applying pulses five times for 99 ms at 0.82 kV using a BTX-830 electroporator. Six hours after the electroporation, grape root extracts or fractions of various concentrations (0.1 ng ~ 10 μg / ml) were treated to the cells and incubated for 72 hours. DMSO was used as a control. Discard the culture medium of the cells treated with the material, wash the well to which the cells are attached with PBS, and then added 20μl cell lysis buffer (cell lysis buffer) and left for 20 minutes in ice.

Thereafter, a dilution of 100-fold dilution of the Renilla Luciferase substrate with Renilla Luciferase buffer was prepared, and 100 μl of the dilution solution was added to each well. The luminescence of Renilla Luciferase was measured at an integration time of 10 seconds. Relative luminescence at the time of grape root extract treatment was calculated based on luminescence at the time of DMSO treatment. Liver cytotoxicity of the grape root fraction was also analyzed in the same manner as in Experimental Example 1. The results are shown in FIGS. 3 to 7.

As shown in Figures 3 to 7, the grape root ethyl acetate fraction, butanol fraction, ethanol extract, hexane fraction, water fraction according to the present invention inhibits the replication of the HCV genome at a concentration of 0.1 ng / ml ~ 10 μg / ml It was confirmed that the activity was excellent, inhibiting the proliferation of HCV and showing no hepatotoxicity.

The replication inhibitory activity of the HCV genome of the grape root methanol extract is shown in FIG. 1, and it was confirmed that the grape root methanol extract not only showed cytotoxicity but also inhibited the replication of the HCV genome.

Experimental Example 3. Determination of Hepatitis C Virus Protein Expression Inhibitory Activity of Grape Root Extract

In order to determine whether the hepatitis C virus genome replication inhibitory activity of the grape root methanol extract confirmed in Experimental Example 2 is shown to inhibit the protein expression of hepatitis C virus, the following experiment was performed.

First, using Bart79I, a subgenomic replicon of the genotype 1b hepatitis C virus disclosed in FIG. 8, to confirm the protein expression level of hepatitis C virus when administration of grape root methanol extract, The amount of fluorescence of GFP (green fluorescent protein) expressed in the genome of was measured by FACS (fluorescent cell analyzer). The results are shown in FIG.

As shown in FIG. 9, when the grape root methanol extract of the present invention was administered at a concentration of 50 ng / ml to 1 μg / ml, it was confirmed that the amount of fluorescence of GFP expressed in hepatocytes was significantly decreased. . Therefore, it was confirmed that the grape root methanol extract has inhibitory activity of viral protein expression through inhibition of genome replication of hepatitis C virus.

In addition, the effect of the grape root methanol extract of the present invention on protein expression of hepatitis C virus was measured by Western blot, and the amount of viral NS5A protein and host cell beta actin protein was measured, and the results are shown in FIG. 10.

As shown in FIG. 10, when the grape root methanol extract of the present invention was administered at a concentration of 50 ng / ml to 1 μg / ml, the protein of hepatitis C virus in hepatitis C infected hepatitis C cell line It was confirmed that the expression decreased concentration-dependently. Therefore, it was confirmed that the grape root methanol extract has an activity of inhibiting the expression of the protein of the virus through the inhibition of genome replication of hepatitis C virus.

Hereinafter, the preparation examples of the pharmaceutical composition including the extract of the present invention will be described, but the present invention is not intended to be limited thereto, but is intended to be described in detail.

Formulation Example 1 Preparation of Pharmaceutical Composition

1-1. Manufacture of powder

Grape root extract or fraction 20 mg

Lactose 100 mg

Talc 10 mg

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

1-2. Manufacture of tablets

Grape root extract or fraction 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

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

1-3. Preparation of Capsules

Grape root extract or fraction 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

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

1-4. Preparation of Injectables

Grape root extract or fraction 10 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na2HPO42H2O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

1-5. Preparation of liquid

Grape root extract or fraction 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding a proper amount of lemon aroma, and then mixing the above components, adding purified water and adjusting the whole to 100 ml by adding purified water and filling into a brown bottle. The solution is prepared by sterilization.

Formulation Example 2 Preparation of Food Composition

2-1. Manufacture of health food

Grape Root Extract or Fraction 100 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

Biotin 10 μg

Nicotinamide 1.7 mg

Folic acid 50 μg

Calcium Pantothenate 0.5 mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

15 mg potassium monophosphate

Dicalcium Phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

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

2-2. Manufacture of health drinks

Grape Root Extract or Fraction 100 mg

15 g of vitamin C

100 g of vitamin E (powder)

Iron lactate 19.75 g

3.5 g of zinc oxide

Nicotinamide 3.5 g

0.2 g of vitamin A

0.25 g of vitamin B1

0.3 g of vitamin B2

Water quantification

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

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

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Grape root extract or fractions thereof according to the present invention has a very low toxicity to hepatocytes, but has an excellent effect of selectively inhibiting the genomic replication of hepatitis C virus, pharmaceutical composition for the prevention or treatment of hepatitis C It can be usefully used as a functional food composition.

Claims (12)

1. Pharmaceutical composition for the prevention or treatment of hepatitis C, comprising grape root (Vitidis Vinferae Radix) extract or a fraction thereof as an active ingredient.
2. [Claim 2] The pharmaceutical composition for preventing or treating hepatitis C according to claim 1, wherein the grape root extract is extracted from grape root with water, alcohol having 1 to 4 carbon atoms or a mixed solvent thereof.
3. The pharmaceutical composition for preventing or treating hepatitis C according to claim 2, wherein the alcohol having 1 to 4 carbon atoms is methanol or ethanol.
4. The method of claim 1, wherein the fraction is ethyl acetate fraction, butanol fraction, hexane fraction, or water fraction obtained by sequentially fractionating grape root extract with ethyl acetate, butanol, hexane, and water. Therapeutic pharmaceutical composition.
5. The pharmaceutical composition for preventing or treating hepatitis C according to claim 1, wherein the grape root extract or fraction has selective inhibitory activity of RNA genome replication of hepatitis C virus.
6. Food composition for the prevention or improvement of hepatitis C containing grape root extract or fractions thereof as an active ingredient.
7. The food composition for preventing or improving hepatitis C according to claim 6, wherein the grape root extract is extracted from grape root with water, alcohol having 1 to 4 carbon atoms or a mixed solvent thereof.
8. 8. The food composition for preventing or improving hepatitis C according to claim 7, wherein the alcohol having 1 to 4 carbon atoms is methanol or ethanol.
9. The method of claim 6, wherein the fraction is ethyl acetate fraction, butanol fraction, hexane fraction or water fraction obtained by sequentially fractionating grape root extract with ethyl acetate, butanol, hexane and water. Food composition for improvement.
10. According to claim 6, The grape root extract or fraction is characterized in that it has a selective inhibitory activity of RNA genome replication of hepatitis C virus, food composition for the prevention or improvement of hepatitis C.
11. A method for preventing or treating hepatitis C, comprising administering to a subject a composition comprising Vitidis Vinferae Radix extract or a fraction thereof as an active ingredient.
12. Use of the composition for the prevention or treatment of hepatitis C of a composition comprising Vitidis Vinferae Radix extract or a fraction thereof as an active ingredient.

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