KR20180136201A - Composition comprising extracts or fraction of Vitidis Vinferae Radix for preventing or treating of noroviral diarrhea as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing or treating norovirus diarrhea comprising an extract or a fraction of Vitis vinifera Radix as an active ingredient. Specifically, the composition according to the present invention has a very low toxicity against hepatocytes and has an excellent effect of selectively inhibiting RNA genome replication of norovirus, thereby being useful for the prevention or treatment of norovirus diarrhea.

Description

[0001] The present invention relates to a composition for prevention or treatment of noroviral diarrhea comprising an extract of a grape vetch or a fraction thereof as an active ingredient. [0002] The present invention relates to a composition for prevention or treatment of noroviral diarrhea,

The present invention relates to a composition for prevention or treatment of noroviral diarrhea comprising an extract of a grape vodka or a fraction thereof as an active ingredient.

Vitrine Vinferae Radix is a herbal medicine that uses the roots of Vitis vinifera and Vitis amurensis, which belong to the Vitaceae (Vitaceae), as it is sun dried or fresh. It is Vitidis Vinferae Radix. "If you drink this water in the month of this month and hiccups stop, And after the pregnancy, when the pregnant woman finishes the meal, she will go down soon after drinking it. "It has been said that it has the efficacy of the traditional ritual, (Small intestine), urine discomfort (discomfort) and has been used to treat. As described above, although various pharmacological effects of grape roots are known (Korean Patent Laid-Open No. 10-2009-0103239), the prevention, improvement, or therapeutic effect of noroviral diarrhea is not known, to be.

Diarrhea, on the other hand, is one of the most frequent diseases in everyday life and has a profound impact on public health. About half of all diarrheas are caused by viruses, and more than 90% of viral diarrhea is caused by norovirus. Norovirus is the most common and clinically important causative microorganism of food intoxication caused by contaminated food.

Norovirus is a non-enveloped, positive-sense RNA virus belonging to the caliciviridae, and the RNA, the genome containing all the genetic information of Norovirus, is approximately 7300 It consists of 7500 nucleic acids. In addition, norovirus is divided into five genotypes (genogroups, GI-GV), in particular, norovirus belonging to genetic groups 1, 2 and 4 (GI, II, and IV) It causes disease.

Norovirus is a very infectious virus that can be infected with only about 10 virus particles, and is infected by ingesting contaminated food through the fecal-to-oral route. Infection with norovirus causes acute gastroenteritis accompanied by severe vomiting and diarrhea. Because of this, Norovirus is also called "stomach flu". In particular, norovirus belonging to genotype 2 (GII.4) causes gastroenteritis caused by norovirus (60-90%). Immuno-competent adults are largely self-resolving at around 72 hours post-Norovirus infection, but elderly, infants, and immuno-compromised patients are often accompanied by vomiting and diarrhea If a gastroenteritis continues and does not receive proper treatment, it will die. Currently, the best way to prevent the spread of Norovirus and reduce infection is to wash your hands frequently and to do personal hygiene thoroughly.

Norovirus infects more than 260 million people worldwide annually, and about 200,000 people die of norovirus-induced gastroenteritis. In the United States, about 21 million cases of gastroenteritis due to norovirus are reported each year, of which about 800 people die from hospitalized and hospitalized more than 70,000. In developing countries, gastroenteritis caused by norovirus causes more than 200,000 deaths annually. Most of the deaths are infants younger than 5 years old. Due to norovirus-induced gastroenteritis, more than $ 4 billion in treatment costs and over $ 60 billion in related social costs are spent annually worldwide.

However, there is no vaccine or antiviral agent that can prevent and treat gastroenteritis caused by norovirus, and currently, the therapy for gastroenteritis caused by norovirus does not provide sufficient water and electrolytes to the light or vein to prevent the patient from dehydrating Rehydration is all. Considering that the social costs incurred by the Norovirus infection are about $ 60 billion, vaccines and treatments for Norovirus to be developed later are expected to be worth more than $ 60 billion.

Norovirus infects host cells using human blood group antigens (HBGAs). Therefore, HBGAs are one of the most important host factors that determine the susceptibility to Norovirus infection. Viral proteins called VPg (virion protein genome-linked) are covalently bound to the 5 'RNA genome terminal of norovirus. This VPg acts as a primer that is essential for RNA genomic replication of Norovirus. Norovirus has three open reading frames (ORFs), and six nonstructural (NS) proteins are expressed in ORF1 (NS1-2: unknown function, NS3NTPase: NTPase, NS4: unknown function, NS5VPg: Vpg protein, NS6Pro: protease, NS7Pol: RNA polymerase) The norovirus capsid proteins VP1 and VP2 are expressed in ORF2 and ORF3, respectively.

Despite decades of enthusiastic research, there have been animal cell lines that can efficiently cultivate and propagate human norovirus (H Norovirus, HNV) Do not. Therefore, it is technically impossible to study the life cycle of whole human norovirus such as invasion of host cell of norovirus, replication of RNA genome in host cell, generation and escape of viral particle in vitro. Therefore, it is urgent to develop a method for the prevention and / or treatment of noroviral diarrhea through studies using the life history of human Norovirus.

Accordingly, the present inventors have continued efforts to develop a novel therapeutic agent for norovirus-induced diarrhea. As a result, the present inventors have found that the extract of the grape vodka or its fractions has a very low toxicity to hepatocytes, and has excellent effect of selectively inhibiting genomic replication of norovirus The present invention has been completed.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition and a health functional food composition for prevention or treatment of noroviral diarrhea comprising an extract of the grape vetch or a fraction thereof as an active ingredient.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for prevention or treatment of noroviral diarrhea comprising an extract of a grapescope or a fraction thereof as an active ingredient.

In one embodiment of the present invention, the grape root extract is extracted with one solvent selected from the group consisting of water, an alcohol having 1 to 4 carbon atoms, and a mixed solvent thereof.

In another embodiment of the present invention, the alcohol having 1 to 4 carbon atoms is methanol or ethanol.

In another embodiment of the present invention, the fraction is one selected from the group consisting of ethyl acetate fraction, butanol fraction, hexane fraction, water fraction, and mixtures thereof.

In another embodiment of the present invention, the extract or fraction of the grape extract is characterized by preventing or treating norovirus-induced diarrhea through selective inhibition of RNA genome replication of norovirus.

The present invention also provides a health functional food composition for prevention or treatment of noroviral diarrhea comprising an extract of a grapescope or a fraction thereof as an active ingredient.

The present invention also provides a method of preventing or treating Noroviral Diarrhea comprising administering the composition to a subject.

The present invention also provides the use of the composition for the prevention or treatment of noroviral diarrhea.

The extract of the grape vodka according to the present invention or the fraction thereof has an extremely low toxicity to hepatocytes and has an excellent effect of selectively inhibiting RNA genome replication of norovirus and is useful for the prevention or treatment of noroviral diarrhea .

Figure 1 is a schematic representation of the structure of the pNV-Neo plasmid used to prepare the HG23 HNV replicon strain.
Figure 2 shows that the expression of NP is increased by genomic cloning of HNV in the HG23 cell line, resulting in increased resistance to G418 antibiotic, and genome replication of HNV reduces genomic replication of HNV, Which is a reduction in resistance.
Figure 3 shows the intracellular location of the RNA genomic cloning complex of HNV in green immunofluorescence using a J2 antibody that specifically binds to the double stranded RNA of HNV.
FIG. 4 shows the results of qRT-PCR to confirm that the amount of HNV RNA decreases in a concentration-dependent manner upon treatment of ribavirin, a genomic replication inhibitor of HNV, on HG23 cells.
FIG. 5 shows colony formation assay that HV23, a genomic replication inhibitor of HNV, is reduced in HNG2 cells treated with HG23 cells, and HNV resistance to G418 antibiotics is decreased.
FIG. 6 is a graph showing the decrease in the expression level of NP protein of HNV in a concentration-dependent manner upon treatment of ribavirin, a genomic replication inhibitor of HNV, with HG23 cells through western bolt.
FIG. 7 is a graph showing the norovirus genome replication inhibitory activity of the extract of the grape vodka or its fractions.
Fig. 8 shows that the grape root fractions hardly have hepatic cytotoxicity.

The present invention provides a composition for the prevention or treatment of noroviral diarrhea comprising the Vitindis Vinferae Radix extract and / 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.

The extract of the grapefruit which is an active ingredient in the composition of the present invention can be obtained by the following method.

First, the vinegars are thoroughly washed with water, dried and then ground. The grapevines may be cultivated or marketed without restriction. A solvent having a volume of 1 to 10 times, preferably 2 to 5 times the weight of the grape muscle weight is added to the crushed grape root so that the grape root is completely immersed and then extracted for 1 to 5 hours, preferably about 2 hours. The extraction solvent may be at least one solvent selected from water, an alcohol having 1 to 4 carbon atoms, and a mixed solvent thereof, preferably methanol or ethanol, but is not limited thereto.

For example, ultrasonic extraction, subcritical extraction, high-temperature extraction, high-pressure extraction, filtration, or a reflux extraction can be used, and ultrasonic extraction can be preferably used. But is not limited thereto.

Thereafter, the extract of the grapefruit extract is separated by centrifugation, and the supernatant is collected and filtered. The filtrate is concentrated under reduced pressure and then lyophilized to obtain powdery grape root extract. In addition, the grape root fractions are obtained by suspending the grape root extract in distilled water, sequentially fractionating the mixture using ethyl acetate, butanol, and hexane, and then extracting the remaining distilled water solubles to obtain ethyl acetate fraction, butanol fraction, hexane fraction, Fractions can be obtained (see Example 1).

The extract of the present invention has a very low toxicity to hepatocytes and has an excellent effect of selectively inhibiting genomic replication of norovirus and thus can be effectively used for the prevention or treatment of noroviral diarrhea.

The composition of the present invention may further contain one or more known active ingredients having an effect of preventing or treating norovirus-induced diarrhea along with the extract of the grapescope or a fraction thereof.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. In addition, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method. Suitable formulations known in the art are preferably those as disclosed in Remington ' s Pharmaceutical Science, recently, Mack Publishing Company, Easton PA.

Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose , Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and suspension include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

In addition, the present invention also provides a method for the prevention or treatment of noroviral diarrhea, comprising the step of administering the composition to a subject, and the use of the composition for the prevention or treatment of noroviral diarrhea.

As used herein, the term " individual " refers to a subject in need of treatment for a disease, and more specifically refers to a human or non-human primate, mouse, rat, dog, cat, horse, Of mammals.

The term " administering " as used herein is meant to provide any desired composition of the invention to a subject in any suitable manner.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and body weight of the individual, the degree of disease, the drug form, the route of administration and the period of time, and can be appropriately selected by those skilled in the art. For the desired effect, the extract of the invention or fraction thereof may be administered in an amount of 0.1 mg / kg to 1000 mg / kg per day, preferably 0.001 to 200 mg / kg, It can also be given in divided doses.

The pharmaceutical composition of the present invention may be administered to a subject in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

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 noroviral diarrhea.

In addition, the present invention provides a health functional food composition for the prevention or amelioration of noroviral diarrhea comprising an extract of a grape vodka or a fraction thereof as an active ingredient. In addition, the grape root extract or its fractions can be added to foods for the purpose of preventing or improving noroviral diarrhea. In the present invention, the health functional food refers to a food having a biological control function such as prevention or improvement of disease, bio-defense, immunity, recovery after disease, suppression of aging, etc., and should be harmless to human body when taken over a long period of time.

When the grapefruit extract of the present invention is used as a food additive, the grapefruit extract may be directly added or used together with other food or food ingredients, and may be appropriately used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, the grape root extract of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight, based on the raw material, when the food or drink is produced. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, 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.

There is no particular limitation on the kind of the food. Examples of the foods to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharine and aspartame, and the like. The ratio 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 may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition of the present invention may comprise flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of such additives is not critical, it is generally 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 embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[ Example ]

Example 1 Preparation of Vineyard Extract and Its Fractions

1-1. Preparation of grape muscle water extract

The vinegars (purchased from Kyungdong Market, Duk-Hyun) were thoroughly cleaned with water, dried and then ground. 100 g of the crushed grapes were immersed in 500 ml of water for 2 hours and extracted with sonication. Then, the resulting extract of the present invention was centrifuged, and the supernatant was separated, collected and filtered. The filtrate was concentrated under reduced pressure and then lyophilized to obtain 14.1 g of powdery grape extract.

1-2. Preparation of Methanol Extract of Vinegar

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

1-3. Preparation of ethanol extract of grape muscle

The ethanol extract of the grape muscle was obtained (8.3 g) in the same manner as in Example 1-1, except that ethanol was used instead of water in Example 1-1.

1-4. Preparation of fractions of the vinegar extract

The ethanol extract of the grape muscle obtained in Example 1-3 was suspended in distilled water and then fractionated with hexane, ethyl acetate, butanol and water sequentially to give a hexane fraction (0.7 g), an ethyl acetate fraction (2.5 g) and a butanol fraction g) and a water fraction (3 g).

Example 2 Production of HG23 Norovirus Replicon Cell Lines

To study norovirus genome replication in vitro, HG23 norovirus replicon cell lines were constructed. The HG23 cell line is the only liver cancer-derived cell line (Huh-7) capable of selectively studying RNA genomic replication of the Norwalk virus belonging to genotype 1 (GI.1) of norovirus. In this Example, pNV-Neo plasmid in which neomycin phosphotransferase (NP) was inserted at the VP1 position was obtained in vitro as shown in Fig. 1 in order to construct the HG23 cell line T7 polymerase was transfected into Huh-7, a hepatoma cell line, and RNAs of HNV transfected into the cells were replicated by NPs to express G418 antibiotics HG23 norovirus replicon cell line was constructed by selecting cells showing resistance. In this example, the amount of RNA of cloned norovirus in HG23 cells was quantified using real-time reverse transcriptase (qRT-PCR).

Example 3. Confirmation of Effect of Ribavirin Treatment on HG23 Cells

In this Example, ribavirin HNV (human norovirus, human norovirus), which has been conventionally used as a therapeutic agent for a non-specific RNA virus disease, Was administered to the HG23 cell line constructed in Example 2 to confirm the antiviral activity against the genomic DNA of the norovirus, as shown in Figs. 4 to 6, to inhibit genomic replication of norovirus in a dose-dependent manner G418 resistance and the expression level of NP protein. In addition, half-maximal effective concentration (EC50), which is half of the genome replication and protein expression of HNV, was found to be approximately 10-20 μM for ribavirin, which is consistent with the EC50 concentration of ribavirin Similar figures.

Example  4. Grape  Extract or its Fraction Norovirus  Measurement of genome replication inhibitory activity

In order to measure the norovirus genome replication inhibitory activity of the methanol extract of the present invention or the ethanol extract of the present invention and its fractions obtained in Example 1, the following experiment was conducted.

The HG23 norovirus replicon cell line prepared in Example 2 was treated with a specific concentration (10 μg / ml) of the extract of the grapescope or its fraction and cultured for 72 hours. DMSO (dimethyl sulfoxide) was used as a control. The amount of RNA of cloned norovirus in HG23 cells was quantified using real-time reverse transcriptase (qRT-PCR). As a result, as shown in Fig. 7, the hexose fraction (Hex) In the treated group, the amount of RNA of norovirus was found to be decreased by 60% or more compared to DMSO. In the case of the grape root butanol fraction (Bu) and the grape root ethyl acetate fraction (EA), it was confirmed that the amount of RNA of norovirus was increased by more than 50%.

Example 5. Hepatic cytotoxicity analysis of hexane fraction of grape root

Cytox cell viability assay (Daeil Lab Service), which is a conventionally known method, was used to examine the hepatocyte toxicity of the fractions of the intracellular hexane obtained in Example 1-4. The same experiment was carried out.

First, 1.7 X 10 ⁴ to 2.0 X 10 ⁴ Huh 7.5 human liver cancer cells per well were added to 96-well plates and cultured in a 37 ° C incubator for 24 hours. The cells were treated with varying concentrations (0.1 ng / ㎖ ~ 10 ㎍ / ㎖) of the vinegar hexane fraction and cultured for 72 hours. The control group was treated with dimethyl sulfoxide (DMSO). After that, the culture medium was discarded, and the cells were washed with PBS. 100 μl of a 1/10 (v / v) dilution prepared by adding a cell culture medium to an EZ-Cytox reagent containing a water-soluble tetrazolium salt was added Lt; / RTI > After the reaction was completed, the absorbance of the cells was measured at a wavelength of 450 nm using a spectrophotometer. The relative absorbance at the time of treatment with the hexane fraction of the vinegar was calculated based on the absorbance at the DMSO treatment of 100.

As a result, as shown in FIG. 8, the grape extract of the present invention exhibited excellent cell survival rate in the range of 0.001 ng / ml to 50 μg / ml, Respectively.

Example 6: Analysis of the effect of concentration of hexane fraction of grape root on the replication of norovirus genome by concentration

The HG23 norovirus replicon cell line prepared in Example 2 was treated with various concentrations (0.001, 0.01, 0.1, 10, and 50 쨉 g / ml) of the grape root hexane fraction and cultured for 72 hours Respectively. DMSO (dimethyl sulfoxide) was used as a control. The amount of RNA of cloned norovirus in HG23 cells was quantified using real-time reverse transcriptase (qRT-PCR). As shown in FIG. 8, The amount of RNA of norovirus was decreased in a concentration-dependent manner compared to DMSO. The concentration of RNA of norovirus by grape hyphae was reduced by more than 50% to about 0.58 ㎍ / ㎖.

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 present invention as defined by the following claims. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (6)

A pharmaceutical composition for prevention or treatment of noroviral diarrhea comprising an extract of a grape vodka or a fraction thereof as an active ingredient.
The method according to claim 1,
Wherein the extract of the grapevine is extracted with one solvent selected from the group consisting of water, an alcohol having 1 to 4 carbon atoms, and a mixed solvent thereof.
3. The method of claim 2,
Wherein the alcohol having 1 to 4 carbon atoms is methanol or ethanol.
The method according to claim 1,
Wherein said fraction is one selected from the group consisting of ethyl acetate fraction, butanol fraction, hexane fraction, water fraction, and mixtures thereof.
The method according to claim 1,
A pharmaceutical composition for preventing or treating a noroviral diarrhea, wherein the extract or fraction of the grape vine is to prevent or treat noroviral diarrhea through selective inhibition of RNA genome replication of norovirus.
A health functional food composition for prevention or improvement of noroviral diarrhea comprising an extract of a grape vodka or a fraction thereof as an active ingredient.

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