KR101643200B1 - A composition comprising mosloflavone compound for preventing and treating the viral disease caused by enterovirus - Google Patents

Abstract

The present invention relates to a composition having an anti-enterovirus, and more particularly, to a composition comprising an extract of gold or a compound isolated therefrom as an active ingredient, which comprises (1) cytotoxicity in vero cells and EV71 , CVB3, CVA16, and cytopathic inhibition experiments; (2) Western blot analysis of the VP protein of enterovirus 71 to confirm antiviral activity against EV71; (3) Analysis of antiviral activity mechanism through replicon analysis using replicon system (pRibFluc-EV71); (4) In vitro tests and in vivo tests in vivo, such as in vivo animal experiments for the determination of antiviral activity against CVB3 virus using BALB / c mice, By confirming that the samples exhibit strong anti-enterovirus activity, the composition can be usefully used as a pharmaceutical composition or health functional food for the prevention and treatment of diseases caused by enteroviruses.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a composition for preventing and treating diseases caused by enteroviruses containing mosloflavone compound as an active ingredient,

The present invention relates to a pharmaceutical composition or health functional food for the prevention and treatment of diseases caused by enteroviruses containing a compound isolated from a golden extract as an active ingredient.

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Enteroviruses are viruses that do not have an envelope of 27 to 30 nm in size. They form a true dodecahedron, and about 68 serotypes have been known to date. It belongs to the Enterovirus genus of Picornaviridae taxonomically. Three genotypes of poliovirus (PV: 1 ~ 3), 23 serotypes of Coxsackievirus A (CVA: 1 ~ 22, 24), six serotypes (ECV: 1 ~ 7, 9, 11 ~ 21, 24 ~ 27, 29 ~ 33) and other human enteroviruses (EV: 68 ~ 116 ) And the like [1-4]. However, recently, researchers enterovirus e nterovirus through the system analysis using the nucleotide sequence of RdRp (RNA dependent RNA polymerase) gene And genotypes of HEV (human enterovirus) A to D [1,5]. There are 11 serotypes of CVA and EV71 in HEV-A, and 38 serotypes of all CVB and ECV, EV69, EV73 and CVA9 in HEV-B. HEV-C contains 11 CVAs. Finally, HEV-D contains only two serotypes, EV68 and EV70 [6].

The genome of Enterovirus s has the form of single stranded positive sense RNA of about 7.2 to 7.5kb in size. It consists of one open reading frame (ORF) and a non-coding region (NCR) that is not expressed as a protein at the 5 'and 3' ends. The ORF is expressed as a polyprotein, consisting of about 2,185 amino acids [7]. This polyprotein is divided into several different proteins by the proteolytic enzyme of the virus. One polyprotein is divided into P1, P2, and P3, which encodes VP4, VP2, VP3, and VP1, which are components of the capsid protein of the virus, in order. The P2 part is 2A ^pro Proteinases and proteins that are not yet known to function correctly are encoded. The P3 part encodes VPg protein and 3C ^pro Protease and RdRp are encoded. The VPg protein acts as a primer on the 3 'side and is involved in the formation of a negative sense strand genome [8]. The 5 'NCR consists of 700 to 800 bp, forming a very complex secondary structure. The function of the secondary structure of these RNAs is firstly provided as a starting point when positive sense RNA is synthesized by RdRp, and secondly, allowing cap-independent protein synthesis [9]. 3'NCR consists of 100-150 bp, and its secondary structure is known to be involved in primer function when negative sense RNA is provided as a template for the RNA genome [10-14].

Enterovirus (Enterovirus) mainly invades infant and childhood during summer season, and may cause severe infection according to the immunological characteristics of the infected person. It is an infectious pathogen that is commonly spread in hygiene-poor environments. The infection pathway is fecal-oral, leading to oral transmission through contaminated water and soil. Fecal viruses from infected people flow through wastewater or wastewater into groundwater, rivers, and seawater, and then again to humans, though rarely through respiratory secretions [15,16]. Enteroviruses are transmitted through the digestive tract and then propagate primarily to the lymph nodes of the larynx or small intestine and then to the organs of the body [17]. Clinical symptoms vary from mild symptoms such as colds to severe paralysis. The incidence of nonpolyposis enterovirus infection is as low as 50% in infants, but in the remaining cases the incidence of upper respiratory infection, digestive symptoms, conjunctivitis, otitis media, skin rash, aseptic meningitis, herpes zoster, . It is rarely reported to cause fatal or complications such as myocarditis, encephalitis, Guillian-Barre syndrome, ataxia, terminal neuritis, transverse myelitis, limb paralysis, diabetes mellitus, and epidemic keratoconjunctivitis [18-22]. The most common symptom of enterovirus is aseptic meningitis, which occurs mainly during the summer, but sporadically occurs in spring, late autumn, and winter, so there is a risk of infection throughout the year. In addition, the main age group can occur in infants and, in some cases, children and elderly [23,24].

  Although antiviral agents with safety, higher efficacy, and lower production cost are currently under development due to increased viral resistance to currently developed antiviral agents, there are still no agents that have therapeutic effects in many viruses [ ]. For this reason, scientists in Asia, Europe and the United States are using data on antiviral agents from traditional medicinal plant-based medicines [26-28]. Medicinal plants often exhibit several antiviral effects, which have low potential for side effects and tolerance and low production costs. Many medicinal plants have already been shown to have potent antiviral effects in viral infected individuals or animals [29,30].

For many viruses belonging to enteroviruses, there is no appropriate mouse model, which is a major obstacle to antiviral research. Recently, a cell receptor for EV71 has been identified, and a transgenic mouse having a human receptor has been developed and studied, but it has not been widely used yet [31,32]. CVB3, on the other hand, is known to infect not only humans but also mice, and is known to infect liver, heart, brain, spinal cord, and pancreas [33, 34]. In the case of CVB3 infection, as described above, it is reported that most people infected with the disease pass through without any specific symptoms, but it has been reported to be associated with the development of some type 1 diabetes [35]. In addition, it has been reported that the Acinar cells and beta cells present in the pancreas are infected with CVB3, and in the mouse model, CVB3-induced pancreatic infections are well known [36, 37].

EV71, CVB3 and CVA16 belonging to the Picornaviridae family are positive-sense, single-stranded RNA viruses [38, 39]. In infants and children, EV71 and CVA16 infections cause early onset infections with mild cold symptoms such as fever, headache, and sore throat. EV71 and CVA16 In addition to causing ulcers and rashes in the hands, feet and mouth within days after infection, respiratory, intestinal, cardiac, and central nervous systems also cause infections [38, 39]. In addition, EV71 can often cause acute relaxation paralysis, such as polio, and encephalitis. CVB3 is known to cause a variety of diseases in children and adolescents, ranging from mild abdominal pain to full pericarditis and myocarditis [40]. However, despite the repeated occurrence of EV71, CVB3 and CV16 in many countries, it is difficult to develop a vaccine or an effective antiviral agent. Therefore, it is urgent to develop an effective antiviral therapeutic agent for treating EV71, CVB3 and CVA16 infection.

The development of antiviral agents against Enterovirus has been continuously studied. Pleconaril has been reported to exhibit antiviral activity by binding to the capsid protein of a virus that binds to receptors in susceptible cells, thereby preventing the virus from entering the cell [41]. Pleconaril has been reported to have a viral suppressive activity in enterovirus infections in 78% of clinically, 92% of virologically, and 88% of experimental cases, but has not been approved by the FDA for its side effects. Most uses are limited to life-threatening situations, and resistant viruses have also been reported [42-44]. Ribavirin, an antiviral agent that exhibits extensive antiviral activity against RNA viruses and DNA viruses, has also been reported to exhibit drug resistance in a variety of viruses [45].

Golden ( Scutellaria baicalensis Georgi ) is a perennial plant belonging to the family Lamiaceae and is widely used as a medicinal plant in China, Japan and Korea. The main efficacy of gold is known to be effective in inflammation, respiratory infections, and gastrointestinal infections [46]. The main ingredients of gold are baicalein, baicalin, wogonin, norwogonin, oroxylin A, sitosterol, mosloflavone, ) Are known [47]. Oroxylin A is known to have many biological activities including anticancer effects [48], antimicrobial effects [49], and cognitive enhancement [50]. The anticancer effect of Oroxylin A is the most actively studied area, and it is known that it has anticancer effect by various mechanisms such as cell suicide [51], metastasis inhibition [52] and cell cycle inhibition [53].

Oroxylin A is a flavone with an O-methyl group found in gold and gold is a useful medicinal plant with bioactive substances such as baicalein and wogonin [54]. Oroxylin A is known to exhibit various pharmacological actions and has been reported to exhibit anti-inflammatory, anti-cancer, and anti-thrombotic effects [55]. Recent studies have also shown that gold extracts, ethyl acetate and chloroform fractions exhibit antiviral activity against influenza virus [56], RSV [57], HBV [58, 59], and HIV-1 [60] Antiviral activity has been reported to be caused by wogonin, oroxylin, or baicalein. In addition, it has recently been reported that water-soluble extracts of gold and baicalein inhibit the replication of dengue virus and exhibit antiviral activity [61, 62].

However, it has been reported that oroxylin A, norwogonin, mosloflavone, and gold extracts and fractions containing them have antiviral activity against enteroviruses none.

In the present invention, oroxylin A, norwogonin, and mosloflavone contained in the gold extract and chloroform fractions and the chloroform fractions exhibit broad antiviral activity against CVB3, CVA16 and EV71 In vitro and in vivo animal experiments such as cell-lesion inhibitory effect and cytotoxicity test in Vero cells. In this study, enterovirus infection treatment of norwogonin, oroxylin A and mosloflavone contained in gold extract, fraction and gold Effect was first identified.

In this study, the gold extracts and the oroxylin A, norwogonin and mosloflavone compounds isolated from the enterovirus (EV71, CVB3, and CVA16) 1) cytotoxicity in Vero cells and antiviral activity tests of EV71, CVB3 and CVA16, experiments for inhibiting cell lesion (Experimental Example 1); (2) Western blot analysis of the VP protein of enterovirus 71 to confirm antiviral activity against EV71 (Experimental Example 2); (3) Experiment for analysis of antiviral activity mechanism by replicon analysis using replicon system (pRibFluc-EV71) (Experimental Example 3); (4) in vitro test and in vivo test such as in vivo animal experiment (Example 4) for the antiviral activity measurement of CVB3 virus using BALB / c mouse The inventors of the present invention confirmed that the samples of the present invention exhibited strong enter enterovirus activity, thereby completing the present invention.

In order to accomplish the above object, the present invention provides a pharmaceutical composition containing, as an active ingredient, a compound selected from the group consisting of gold extract or norwogonin, oroxylin A, and mosloflavone isolated therefrom The present invention provides a pharmaceutical composition for preventing and treating infectious diseases caused by enteroviruses.

The present invention also relates to a pharmaceutical composition comprising a combination of a compound selected from the group consisting of gold extract or isolated therefrom, such as norwogonin, oroxylin A, and mosloflavone, A pharmaceutical composition for the prevention and treatment of infectious diseases caused by enteroviruses.

The present invention also provides an anti-enteroviral agent containing, as an active ingredient, a compound selected from the group consisting of a gold extract or isolated therefrom, norwogonin, oroxylin A, and mosloflavone.

The present invention relates to a method for the treatment of an infectious disease caused by an enterovirus comprising, as an active ingredient, a compound selected from the group consisting of gold extracts or isolated from them, norwogonin, oroxylin A, and mosloflavone. And a health functional food for prevention and improvement.

The gold extract as defined herein is characterized by being a crude extract, a polar solvent-soluble extract or a non-polar solvent-soluble extract of a plant including golden stem, leaf and root stem.

The crude extract as defined herein may be dissolved in a solvent selected from water including purified water, a lower alcohol having 1 to 4 carbon atoms such as methanol, ethanol, and butanol, or a mixed solvent thereof, preferably methanol or water and a methanol mixed solvent, And is an extract which is soluble in methanol.

The nonpolar solvent-soluble extract fractions defined herein contain extract fractions soluble in nonpolar solvents purified from the crude extract of the present invention in hexane, methylene chloride, chloroform, or ethyl acetate, preferably hexane, or chloroform solvent alone do.

The polar solvent-soluble extract as defined herein is prepared by removing the non-polar solvent-soluble fractions from the crude extract and washing with water, methanol, butanol or a mixed solvent thereof, preferably water or butanol, more preferably, And one extracted fraction.

Enteroviruses as defined herein include three serotypes of Poliovirus (PV: 1-3); Coxsackievirus A group of 23 serotypes (CVA: 1 to 22, 24); Six serotypes of Coxsackievirus B (CVB: 1 to 6); A serotype selected from the group consisting of 28 serotypes of Echovirus (ECV: 1-7, 9, 11-21, 24-27, 29-33) and other human enteroviruses (EV: 68-111) Is characterized by having an EV71, CVB3, or CVA16 serotype.

The diseases caused by enteroviruses as defined herein include those caused by enteroviruses, such as, but not limited to, those caused by atopic diseases, gastrointestinal tract infections, gastrointestinal symptoms, conjunctivitis, otitis media, skin rash, aseptic meningitis, herpetic sinusitis, Acute atonic paralysis such as Guillian-Barre syndrome, ataxia, terminal neuritis, transverse myelitis, limb paralysis, diabetes mellitus, epidemic hemorrhagic keratoconjunctivitis, ulcers, rashes, polio, pericarditis, type 1 diabetes, or pancreatic infections, preferably Include conjunctivitis, conjunctivitis, aseptic meningitis, herpetic sinusitis, myocarditis, encephalitis, type 1 diabetes, or pancreatic infections.

Existing anti-enterovirus agents as defined herein are selected from the group consisting of Pleconaril, Rupintrivir, Oseltamivir (Tamiflu), Zanamivir, Relenza, Peramivir, Amantadine Preferably selected from the group consisting of Pleconaril, Rupintrivir or Rivabirin, which is an antiviral selected from Amantadine, Symmetrel, Rimantadine, Rivabirin, or Taribavirin. do.

The disease is characterized in that it occurs in mammals including humans.

The composition comprising the gold extract of the present invention or a compound isolated therefrom as an active ingredient comprises 0.1 to 50% by weight of the extract or the compound isolated therefrom based on the total weight of the composition.

The compounds of the present invention may be prepared into pharmaceutically acceptable salts and solvates by methods conventional in the art.

Pharmaceutically acceptable salts as defined herein are acid addition salts formed by free acids. The acid addition salt is prepared by a conventional method, for example, by dissolving the compound in an excess amount of an acid aqueous solution, and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The same molar amount of the compound and the acid or alcohol (e.g., glycol monomethyl ether) in water may be heated and then the mixture may be evaporated to dryness, or the precipitated salt may be filtered by suction.

As the free acid, organic acids and inorganic acids can be used. As the inorganic acids, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used. Examples of the organic acids include methanesulfonic acid, p -toluenesulfonic acid, acetic acid, trifluoroacetic acid Citric acid, lactic acid, glycollic acid, gluconic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid and hydroiodic acid can be used.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving a compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-soluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt in particular, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

Pharmaceutically acceptable salts of the compounds of the present invention include, unless otherwise indicated, salts of acidic or basic groups that may be present in the compounds of the present invention. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of hydroxy groups, and other pharmaceutically acceptable salts of amino groups include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate rate, methane sulfonate (mesylate) and p - toluene sulfonate (tosylate) and a salt, the salt manufacturing method or manufacturing process known in the art ≪ / RTI >

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

The extracts and compounds of the present invention can be obtained by the following production methods.

For example, the present invention will be described in detail below.

The gold extract of the present invention can be prepared as follows. After drying and finishing the dried gold outpoule, it is mixed with a solvent selected from water, a low alcohol having 1 to 4 carbon atoms such as methanol, ethanol, and butanol, or a mixed solvent thereof, preferably water, methanol, ethanol and a mixed solvent thereof And then subjected to ultrasonic extraction, hot water extraction, room temperature extraction or reflux extraction, preferably room temperature extraction, at about 30 ° C to 150 ° C, preferably at room temperature for about 12 hours to 30 days, preferably for 1 day to 7 days, To 20 times, preferably 2 to 10 times. The extract obtained by filtration, concentration under reduced pressure, and dried can be used to obtain the crude extract of the present invention.

The polar solvent or the non-polar solvent soluble extract of the present invention may further contain about 0.0005 to 5 times, preferably 0.05 to 0.5 times the volume (v / w%) of the crude extract, preferably 30 to 90% ), Followed by fractionation using n-hexane, methylene chloride, ethyl acetate and butanol to obtain non-polar solvent-soluble extract fractions, which are dissolved in a nonpolar solvent such as n-hexane, methylene chloride or ethyl acetate; And polar solvent-soluble extract fractions soluble in polar solvents such as butanol and water can be obtained.

The non-polar solvent-soluble fraction extracted in the non-polar solvent, such as n-hexane, methylene chloride or ethyl acetate, obtained in the above, is dissolved in hexane and methylene chloride mixed solvent in a flash column The purification method using chromatography such as chromatography, RP C18 column chromatography or silica gel open column chromatography may be repeated several times to selectively purify and obtain reynoudiol, which is a compound of the present invention.

(1) cytotoxicity in vero cells and antiviral activity tests of EV71, CVB3 and CVA16, experiments for inhibition of cell lesion (Experimental Example 1), and ; (2) Western blot analysis of the VP protein of enterovirus 71 to confirm antiviral activity against EV71 (Experimental Example 2); (3) Analysis of antiviral activity mechanism through replicon analysis using replicon system (pRIBFluc-EV71) (Experimental Example 3); (4) in vitro test and in vivo test such as in vivo animal experiment (Example 4) for the antiviral activity measurement of CVB3 virus using BALB / c mouse Through confirming that the samples of the present invention exhibit strong anti-enteroviral activity, it is confirmed that the composition is useful as a pharmaceutical composition or health functional food for the prevention and treatment of diseases caused by enteroviruses.

Accordingly, the present invention provides a pharmaceutical composition and a health functional food for the prevention and treatment of diseases caused by enterovirus containing the gold extract or the compound isolated therefrom as an active ingredient.

The pharmaceutical composition of the present invention contains 0.1 to 50% by weight of the above extract or compound, based on the total weight of the composition.

In addition, the stem, leaf and stem of gold have been used for a long time as a medicinal herb or as a herbal medicine, and the compounds isolated from the gold extract of the present invention are also free from toxicity and side effects.

Excipients and diluents conventionally used in the preparation of pharmaceutical compositions containing the extracts or compounds of the present invention.

The pharmaceutical dosage forms of the extracts or compounds of the present invention may be used in the form of their pharmaceutically acceptable salts and may be used alone or in combination with other pharmacologically active compounds as well as in suitable aggregates.

The composition containing the extract or the compound according to the present invention can be administered orally or parenterally in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like, external preparation, Can be used. Examples of carriers, excipients and diluents that may be contained in the composition containing the extract or compound 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. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is 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. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol gelatin and the like can be used.

The preferred dose of the extract or compound of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract or the compound of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The extract or compound of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The present invention also relates to a method for the treatment or prevention of diseases caused by enteroviruses containing, as an active ingredient, a compound selected from the group consisting of gold extracts or isolated therefrom, norwogonin, oroxylin A, and mosloflavone A health functional food for preventing and improving diseases is provided.

Examples of foods to which the extract or compound of the present invention can be added include various foods, beverages, gums, tea, vitamin complexes, and health functional foods.

It may also be added to foods or beverages for the purpose of preventing diseases caused by enteroviruses. In this case, the amount of the extract in the food or drink may be 0.01 to 15% by weight of the total food, and the health beverage composition may be added in a proportion of 0.02 to 5 g, preferably 0.3 to 1 g, .

The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids.

The health functional beverage composition of the present invention is not particularly limited to the ingredients other than the above-mentioned extract or compound as an essential ingredient in the indicated ratios and may contain various flavors or natural carbohydrates as an additional ingredient have. 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, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. As natural flavors other than those described above, natural flavors (such as tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin)) and synthetic flavors (saccharin, aspartame, etc.) have. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

The present invention also relates to a method for the treatment of diseases caused by enterovirus, comprising the step of administering a compound selected from the group consisting of a gold extract for the improvement of diseases caused by enterovirus or isolated therefrom, norwogonin, oroxylin A, and mosloflavone Provide health supplements containing the main ingredient.

The present invention also relates to a method for the treatment of diseases caused by enterovirus, comprising the step of administering a compound selected from the group consisting of a gold extract for the improvement of diseases caused by enterovirus or isolated therefrom, norwogonin, oroxylin A, and mosloflavone And a food or food additive containing the main ingredient.

In addition, the health functional food may further include food additives, and the suitability of the food functional food as a "food additive" Standards and standards.

Examples of the products listed in the above-mentioned "food additives" include natural products such as ketones, chemical products such as glycine, potassium citrate, nicotinic acid and cinnamic acid, coloring matter, licorice extract, crystalline cellulose, guar gum, Sodium laurate, sodium glutamate preparation, noodles-added alkaline agent, preservative agent, tar pigment preparation and the like.

 Examples of the functional food containing the extract or the compound of the present invention include confectionery ice cream such as bread, rice cake, dried fruit, candy, chocolate, chewing gum and confectionery, ice cream such as ice cream, ice cream powder, low- Dairy products such as cracked milk, processed oil, goat milk, fermented oil, butter oil, concentrated oil, yogurt cream, butter oil, natural cheese, processed cheese, powdered milk, milk products, meat products such as egg products and hamburgers Fish noodles such as ham, sausage, and bacon; noodle products such as ramen noodles, noodle noodles, fresh noodles, noodle noodles, luxury noodles, improved sleep noodles, frozen noodles, noodles such as pasta, Such as beverages, bean paste, red pepper paste, chunks, chonggukjang, mixed berries, vinegar, sauces, tomato ketchup, curry, dressing, etc. US food but are margarine, shortening and pizza, without being limited thereto.

The health functional beverage composition of the present invention is not particularly limited to the ingredients other than the above-mentioned extract or compound as an essential ingredient in the indicated ratios and may contain various flavors or natural carbohydrates as an additional ingredient have. Examples of the above-mentioned natural carbohydrates include monosaccharides (e.g., glucose, fructose, etc.); Disaccharide, (e.g., maltose, sucrose, etc.); And polysaccharides (for example, dextrin, cyclodextrin and the like), and sugar alcohols such as xylitol, sorbitol and erythritol. As natural flavors other than those described above, natural flavors (such as tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin)) and synthetic flavors (saccharin, aspartame, etc.) have. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the extract or the compound of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, And salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the extract or the compound 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 so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

As mentioned above, the gold extract of the present invention and the compounds isolated therefrom are (1) cytotoxicity in vero cells and antiviral activity tests of EV71, CVB3 and CVA16, One); (2) Western blot analysis of the VP protein of enterovirus 71 to confirm antiviral activity against EV71 (Experimental Example 2); (3) Analysis of antiviral activity mechanism through replicon analysis using replicon system (pRIBFluc-EV71) (Experimental Example 3); (4) in vitro test and in vivo test such as in vivo animal experiment (Example 4) for the antiviral activity measurement of CVB3 virus using BALB / c mouse By confirming that the samples of the present invention exhibit strong anti-enterovirus activity, the composition is useful as a pharmaceutical composition or health functional food for the prevention and treatment of diseases caused by enteroviruses.

1 is a graph showing the effect of a sample of the present invention on cytotoxicity caused by EV71 infection, wherein (A) noninfected cells, (B) norwogonin noninfected cells treated with norwogonin, ; (C) nonoxed cells treated with oroxylin A; (D) noninfected cells treated with oroxylin A; (D) noninfected cells treated with mosloflavone; (E) ribavirin treated with ribavirin; (EV71-infected cells treated with norwogonin) (EV71-infected cells) (EV71-infected cells) (EV71-infected cells treated with norwogonin) (I) EV71-infected cells treated with mosloflavone; (J) EV71-infected cells treated with ribavirin);
FIG. 2 shows the effect of the present invention on cytotoxicity by CVB3 infection (A) noninfected cells; (B) noninfected cells treated with norwogonin (B) ; (C) nonoxed cells treated with oroxylin A; (D) noninfected cells treated with oroxylin A; (D) noninfected cells treated with mosloflavone; (E) ribavirin treated with ribavirin; CB3-infected cells (CB3-infected cells); (G) norwogonin CB3-infected cells treated with norwogonin; (H) oroxylin A sample CB3-infected cells (J) CB3-infected cells treated with ribavirin, respectively); (iii) moxifloxacin, oroxylin A; (i) mosloflavone CB3-infected cells treated with mosloflavone;
FIG. 3 is a graph showing the effect of the present invention on cytotoxicity caused by CVA16 infection (A) noninfected cells; (B) noninfected cells treated with norwogonin (B) ; (C) nonoxed cells treated with oroxylin A; (D) noninfected cells treated with oroxylin A; (D) noninfected cells treated with mosloflavone; (E) ribavirin treated with ribavirin; (F) CVA16-infected cells (G) norwogonin sample CVA16-infected cells treated with norwogonin; (H) oroxylin A sample CVA16-infected cells (J) CVA16-infected cells treated with ribavirin, respectively); (iv) moxifloxacin, oroxylin A; (I) mosloflavone sample CVA16-infected cells treated with mosloflavone;
Figure 4 shows the antiviral activity of the sample of the present invention against EV71 (the results are expressed as the mean ± SD of the percentage values of the independent triplicate experiments);
Figure 5 shows the antiviral activity of the samples of the present invention against CVB3 (the results are expressed as the mean ± SD of percentages of independent triplicate experiments);
Figure 6 is a graph depicting the antiviral activity of the samples of the invention against CVA16 (the results are expressed as the mean ± SD of percentages of independent triplicate experiments);
7 is a graph showing the effect of the sample of the present invention on the expression of EV71 virus Capside protein;
Figure 8 shows the effect of the sample of the present invention on EV71 viral replication and translation;
FIG. 9 is a graph showing changes in mouse body weight by administration of gold extract and oroxylin A in CVB3-infected BALB / c mice;
FIG. 10 shows pancreatic tissue analysis by gold extract and oroxylin A administration in CVB3-infected BALB / c mice; (A) A: Non-infected group; B: Virus infected group; C: Golden methanol extract treatment Virus infected group; D: Oroxylin A Sample treatment Virus infection group (B) Number of acini cells in the pancreas.

Below, The present invention will be described in detail by the following examples and experimental examples.

However, the following examples and experimental examples are illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

Example 1. Gold extract and fraction

1-1. Preparation of crude extract of gold

6.0 L of methanol was added to 1.2 kg of dry gold purchased from Daelim Construction Materials Co., Ltd. located in Cheongju City, and the mixture was immersed at room temperature for 24 hours. The mixture was concentrated using a vacuum concentrator to obtain 66.3 g of methanol extract (hereinafter referred to as SBM , And the antiviral activity against CVB3 was measured.

1-2. Fractions of polar solvent and non-polar solvent soluble extract

The methanol extract (66.3 g) obtained in Example 1-1 was suspended in distilled water and purified by using chloroform, ethyl acetate and n-butanol (BuOH) according to a conventional fractionation method (15.6 g, hereinafter referred to as SBC), ethyl acetate soluble fraction (18.2 g, hereinafter referred to as SBE) and n-BuOH soluble fraction (13.6 g, hereinafter referred to as SBB) ), And then fractionated into a water fraction (hereinafter referred to as SBW). The antiviral activity of each fraction was examined to find that the chloroform layer showed significant antiviral activity (Table 1).

Antiviral activity of S. baicalensis Georgi against CVB3 in vero cell Coxsackievirus B3 Compound CC ₅₀ ^a IC ₅₀ ^b TI ^c Methanol (SBM) 62.5 9.53 + - 0.85 1.6 Chloroform (SBC) 37.5 9.76 + 0.26 2.4 Ethyl acetate (SBE) 91.6 ND ^d 1.8 Butanol (SBB) 113.5 ND ^d Water ND ^d ND ^d Results are presented as the mean IC ₅₀ values ± SD obtained from three independent experiments carried out in triplicate.
^a Concentration required to reduce cell growth by 50% (μg / mL).
^b Concentration required to inhibit virus-induced CPE by 50% (μg / mL).
^c Therapeutic index = CC ₅₀ / IC ₅₀
^d Not determined

1-3. Isolation of active compounds

The chloroform layer exhibiting antiviral activity in Example 1-2 was subjected to C18 silica gel column chromatography (C-18 silica gel column, adsorption chromatography, 2.5 x 25 cm) with methanol and water at 3: 7, 4: 6, 6: 4, 8: 2, and 10: 0 ratios were separated into 11 fractions using two 500 ml elution solvents. Fractions 6 to 13 were isolated and purified by column chromatography (2.5 × 170 cm) on a sephadex LH-20 column (25-100 μm, Sigma-Aldrich, Steinheim, Germany) using 100% methanol. The structures were identified by norwogornin (Table 2), oroxylin A (Table 3), and mosloflavone (Table 4), respectively, by comparing ESI-MS, ¹ HNMR and ¹³ C NMR analysis [63-65]

¹ H-NMR and ¹³ C-NMR chemical shifts of norwogonin Position ¹³ C-NMR ¹ H-NMR 2 163.0 3 104.6 6.93 (S) 4 182.2 5 153.1 6 98.8 6.34 (s) 7 145.6 8 125.1 9 153.7 10 103.9 One 130.9 2 126.5 8.16 (dd, J = 6.0,2.0) 3 129.0 7.59 (m) 4 131.9 7.59 (m) 5 129.0 7.59 (m) 6 126.5 8.16 (dd, J = 6.0,2.0) -OH 8.83 (s) -OH 10.54 (s) -OH 12.27 (s)

Norwogornin's  rescue

¹ H-NMR and ¹³ C-NMR chemical shifts of oroxyline Position ¹³ C-NMR ¹ H-NMR 2 163.2 3 104.6 6.95 (s) 4 182.2 5 152.7 6 130.7 7 157.6 8 94.4 6.63 (s) 9 152.5 10 104.3 One 131.5 2 126.4 8.05 (dd, J = 6.4, 1.6) 3 129.1 7.58 (m) 4 132.0 7.58 (m) 5 129.1 7.58 (m) 6 126.4 8.05 (dd, J = 6.4, 1.6) -OCH3 59.934 3.76 (s) -OH 10.79 (s) -OH 12.92 s)

Oroxylin  Structure of A

¹ H-NMR and ¹³ C-NMR chemical shifts of mosloflavone Position ¹³ C-NMR ¹ H-NMR 2 163.4 3 104.9 7.02 (s) 4 182.3 5 152.0 6 131.9 7 158.8 8 91.7 6.95 (s) 9 152.7 10 105.3 One 130.6 2 126.4 8.09 (dd, J = 6.8,1.2) 3 129.1 7.59 (m) 4 132.1 7.59 (m) 5 129.1 7.59 (m) 6 126.4 8.09 (dd, J = 6.8,1.2) -OCH3 60.0 3.74 (s) -OCH3 56.4 3.93 (s) -OH 12.76 (s)

Mosloflavone  rescue

Antiviral activity of Norwogonin, Oroxylin A and Mosloflavone against EV71, CVB3 and CVA16 in vero cell Enterovirus 71 Coxsackievirus B3 Coxsackievirus A16- Compound CC ₅₀ ^a IC ₅₀ ^b TI ^c CC ₅₀ ^a IC ₅₀ ^b TI ^c CC ₅₀ ^a IC ₅₀ ^b TI ^c Norwogonin > 50 31.9 ± 6.23 1.6 > 50 13.5 ± 9.83 3.7 > 50 ND ^d - Oroxylin > 50 20.6 ± 1.93 2.4 > 50 3.17 ± 1.19 16 > 50 17.5 ± 13.7 2.7 Mosloflavone > 50 27.8 ± 6.67 1.8 > 50 3.92 ± 2.15 13 > 50 ND ^d - Ribavirin > 50 ND ^d > 50 ND ^d > 50 ND ^d - Results are presented as the mean IC ₅₀ values ± SD obtained from three independent experiments carried out in triplicate.
^a Concentration required to reduce cell growth by 50% (μg / mL).
^b Concentration required to inhibit virus-induced CPE by 50% (μg / mL).
^c Therapeutic index = CC ₅₀ / IC ₅₀
^d Not determined

Reference Example 1. Viruses, cell lines and reagents

Enterovirus was obtained from the Microbiology Section of the Institute of Health and Environment, Chungnam Province and was propagated in vero cells (CCL-81, ATCC) at 37 ℃. Vero cells were maintained in DMEM medium supplemented with 10% FBS and 0.01% antibiotics. Antibiotics, trypsin EDTA (15400-054), FBS (16000-044) and DMEM media (12491-015) were purchased from Gibco. Tissue culture dish (353003) was purchased from Falcon and sulforhodamine B (SRB (S9012-25G)) was purchased from SigmaAldrich. Ribavirin (R0182) was purchased from DUCHEFA (Netherlands). DMSO was used as a solution for the antiviral compound and diluted in DMEM media. The final concentration of DMSO was used as the maximum concentration of 0.1%, which had no effect on cell culture. 0.1% DMSO was used as a cell control.

Experimental Example 1. Antiviral activity test

In order to confirm the antiviral activity and cytotoxicity of the sample of the above example by the SRB assay for the ability to inhibit the cell lesion caused by the virus, experiments were conducted as described below by using the method described in the literature [66, 67 ].

1-1. Antiviral activity test

Namely, each virus-susceptible cell was prepared in each well of a 96-well culture plate (Nunc, Denmark) in a number of 2 × 10 ⁴ . After 24 h, each cell was grown in a 96-well culture plate at 90%. Media was removed from each well and each cell was washed once with PBS. After that, Norwogonin, oroxylin A, and 10 μL of mosloflavone were added to 90 μL of DMEM containing 1% FBS and each virus. Antiviral activity of Norwogonin, oroxylin A, and mosloflavone was determined at four concentrations of 0.4 μg / mL, 2 μg / mL, 10 μg / mL, and 50 μg / mL.

Four wells were used as the cell control without virus treatment and the other four wells were used as the cell control without virus and compound treatment. A CO ₂ incubator (VS-9108MS, Vision, Korea ) a phase contrast microscope in the form of after incubation for 2 days by 37 ℃, conditions of 5% CO ₂ cells; in (Axiovert 10 Zeiss, Wetzlar, Germany ) with 4 X 10 magnification After observation, images were recorded.

1-2. Cytotoxicity test

Each cell cultured in 96-well plates was treated with four concentrations of norwogonin, oroxylin A, and mosloflavone at concentrations of 0.4 μg / mL, 2 μg / mL, 10 μg / mL, and 50 μg / mL, After incubation at 37 ° C for 2 days in a CO ₂ incubator, cytotoxicity was evaluated by SRB assay.

1-3. Experiment result

Antiviral Activity and Cytotoxicity of Gold Methanol Extracts and Fractions on CVB3

The methanol extracts were fractionated into chloroform, ethyl acetate, butanol and water. The anti - viral activity of CVB3 was examined by using golden methanol extract and each fraction. The concentrations of 50 μg / mL, 10 μg / mL, 2 μg / mL and 0.4 μg / mL of gold methanol extract and each fraction were used. The cytotoxicity of each extract and fraction layer was cytotoxic in all the fraction layers except the water fraction layer at the maximum drug concentration of 50 μg / mL used in the experiment. However, at the concentration of 10 μg / mL, neither the extract nor the respective fraction layers showed cytotoxicity. Antiviral activity The antiviral activity of CVB3 was about 50% in the golden methanol extract and chloroform fraction at the concentration of 10 μg / mL, but did not show any antiviral activity in the ethyl acetate, butanol and water fractions. The IC ₅₀ (50% inhibitory concentration) values of the golden methanol extract and the chloroform fraction of CVB3 were 9.35 μg / mL and 9.76 μg / mL, respectively (Table 1).

Antiviral activity and cytotoxicity of norwogonin, oroxylin A and mosloflavone against EV71, CVB3, and CVA16

Norwogonin, oroxylin A and mosloflavone were measured for cytotoxicity and antiviral activities of EV71, CVB3 and CVA16. The CC ₅₀ of norwogonin, oroxylin A, and mosloflavone were not shown at the maximum concentration of 50 μg / mL in the Vero cells used. The IC _{50 values} showing antiviral effects that inhibit viral activity by 50% were 31.9 μg / mL for norwogonin, 20.6 μg / mL for oroxylin A, 27.8 μg / mL for mosloflavone, and 13.5 μg / mL, oroxylin A and mosloflavone were 3.17 μg / mL and 3.92 μg / mL, respectively. However, for the CVA16, oroxylin A showed an IC ₅₀ value of 17.5 μg / mL, while norwogonin and mosloflavone showed no antiviral activity of more than 50%. In addition, ribavirin used as a positive control did not show more than 50% antiviral activity against all viruses of EV71, CVB3, and CVA16 (Table 4).

Cellular lesion inhibition effect of Norwogonin, oroxylin A and mosloflavone on EV71, CVB3, and CVA16

Vero cells were infected with EV71 (Fig. 1), CVB3 (Fig. 2), and CVA16 (Fig. 3). After 2 days, cell lesions were imaged using a microscope. (FIG. 1A, FIG. 2A, and FIG. 3A) and the group treated with 50 μg / mL norwogonin without virus infection (FIG. 4B, FIG. 5B and FIG. 6B), 50 μg of oroxylin A (Fig. 1C, Fig. 2C and Fig. 3C) treated with 50 袖 g / mL of mosloflavone and the group treated with 50 袖 g / mL ribavirin And Fig. 3E) showed normal cell shapes as in the normal control group.

As a result, Norwogonin, oroxylin A, mosloflavone and ribavirin were not cytotoxic. (Fig. 1F, Fig. 2F and Fig. 3F) infected with EV71, CVB3 and CVA16 and treated with 50 μg / ml of norwogonin (Fig. 1H, Fig. 2H and Fig. 3H) treated with 50 μg / mL of oroxylin A and 50 μg / mL of ribavirin treated with 50 μg / mL of mosloflavone (Fig. 1J, Fig. 2J and Fig. 3J), the infection control group and the ribavirin treatment group showed cell lesions caused by each virus. Norwogonin, oroxylin A, and mosloflavone inhibited cell lesions in both EV71 and CVB3, and showed normal cell morphology.

However, for CVA16, oroxylin A inhibited cell lesion, but norwogonin and mosloflavone did not inhibit cell lesion. Each group was imaged and cell viability was measured using a microplate reader. Norwogonin, oroxylin A, and mosloflavone showed more than 60% cell viability for EV71 (FIG. 4), and each compound showed more than 50% cell viability for CVB3 (FIG. 5). In CVA16, however, only oroxylin A showed cell viability of more than 50% (Fig. 6).

Experimental Example 2. VP-Western blot analysis test

In order to confirm the antiviral activity of norwogonin, oroxylin A, and mosloflavone against EV71, Western blot analysis of the VP protein of enterovirus 71 described in the literature was applied as follows [68].

2-1. Experimental course

Vero cells were cultured in 6-well culture plates at 5 × 10 ⁵ cells per well. After 24 hours, the culture solution was removed and washed with PBS. Then, 100 μL of norwogonin, oroxylin A, and mosloflavone and ribavirin were added to 900 μL of virus culture medium containing 1% FBS at a concentration of 50 μg / mL. Cells were then lysed in RIPA lysis buffer (Cat. No. 89900, Pierce) at 37 ° C in a 5% CO ₂ incubator for 48 h. The dissolved cells were centrifuged to separate only the supernatant containing the protein. The protein-containing supernatant was heated at 100 ° C. for 10 minutes and then electrophoresed at 100 V for 1 hour using 12% acrylamide gel (Cat No. 456-1043, BIO-RAD). The protein in acrylamide gel was then transferred to iBlotTransfer Stack, PVDF, and regular-size (IB401001, Invitrogen) at 20V for 7 min using a device (iBlotGel Transfer Device, Invitrogen, Carlsbad, CA). The membrane was incubated in 5% skim milk (232100, Difco) for 1 hour at room temperature and then washed three times with PBST (Phosphate buffer saline Tween-20). The primary antibody diluted 1: 1000 in 5% skim milk was reacted with the mouse anti-enterovirus 71 monoclonal antibody (MAB 979, Millipore) for 2 hours at room temperature and then washed three times with PBST gave. In this experiment, α-tubulin, which was used as a control, was subjected to the same method using mouse monoclonal IgG1 (SC-32293, Santa Cruz) as a primary antibody. The secondary antibody to identify the VP protein of Enterovirus 71 and α-Tubulin as the control group was polyclonal goat anti-mouse IgG (H + L) HRP (SA001-500, GenDEPOT). polyclonal goat anti-mouse IgG (H + L) HRP was diluted in 5% skim milk at a ratio of 1: 5000 and reacted at room temperature for 1 hour. Then, the cells were washed 3 times with PBST, Imaging System, BIO-RAD, Hercules, CA, USA).

2-2. Inhibitory Effect of Norwogonin, oroxylin A and mosloflavone on EV71 VP Protein Expression

Antiviral activity of norwogonin, oroxylin A and mosloflavone against EV71 was confirmed by Western blot analysis. After 48 hours of EV71 infection, the VP protein of EV71 was identified. Norwogonin, oroxylin A and mosloflavone inhibited the expression of the respective viral VP proteins 48 hours after EV71 infection. However, ribavirin used as a control agent did not inhibit the expression of each viral VP protein at a concentration of 50 μg / mL (FIG. 7).

Finally, norwogonin, oroxylin A and mosloflavone showed antiviral activity against EV71, CVB3 and CVA16 through CPE inhibition experiments and western blot.

Experimental Example 3. Antiviral Activity Mechanism Analysis Test

To analyze the antiviral activity mechanism of norwogonin, oroxylin A and mosloflavone, replicon analysis method described in the literature was applied using the replicon system (pRibFluc-EV71) provided by Frank JM van Kuppeveld group as follows [69 ].

3-1. Experimental Process

pRibFluc-EV71 replaces the P1 capside coding region of EV71 with a firefly luciferase gene, which can clearly observe the effects of replication and translation except entry, penetration, uncoating, assembly, and release in the virus life cycle system [69].

Plasmid pRibFluc-EV71 was linearized with MluI enzyme (1071A, TaKaRa) and in vitro transcription was performed with T7 RNA polymerase (P1280, promega). Transcribed RNA was transfected into vero cells by 2 μg of x-treme (04476093001, Roche) and treated with norwogonin, oroxylin A, and mosloflavone at a concentration of 10 μg / mL for 8 hours. Then, one-glo (E6120, Promega) is used to measure firefly luciferase activity.

3-2. Effects of Norwogonin, oroxylin A and mosloflavone on EV71 replication and traslation

The replication and traslation inhibitory effects of Norwogonin, oroxylin A and mosloflavone on EV71 were analyzed using a replicon system. The firefly luciferase activity was confirmed 8 hours after trasfection of pRibFluc-EV71. Norwogonin inhibited firefly luciferase activity by about 50% after 8 h, while oroxylin A and mosloflavone did not inhibit firefly luciferase activity (Fig. 8). On the other hand, rupintrivir, known as an enterovirus protease inhibitor, inhibited more than 95% of firefly luciferase activity under the same conditions.

To investigate the activity of norwogonin, oroxylin A and mosloflavone, we investigated the activation mechanism of these compounds using EV71 replicon system (pRIBFluc-EV71) in cells. As a result, norwogonin was involved in replication or translation of virus Indicating that it exhibits antiviral activity. However, considering that these effects are relatively weak compared to the antiviral effects confirmed in viral infected vero cells, it is not possible to exclude the possibility that norwogonin exhibits antiviral effects through other mechanisms besides replication / translation. Whereas oroxylin A and mosloflavone were not involved in the replication or translation of the virus.

Experimental Example 4: In vivo antiviral activity test

In order to confirm the antiviral activity against the CVB3 virus of the above-mentioned samples in vivo in an animal experiment, the following experiment was conducted by applying the method described in the literature [70]. In this experiment, oroxylin A and gold methoanl extracts, which were found to have the highest antiviral effect among norwogonin, oroxylin A and mosloflavone, were used in cell experiments.

4-1. Experimental Process

In order to confirm the antiviral activity of the golden methanol extract and oroxylin A against CVB3 in vivo , the experiment was carried out using BALB / c mouse.

Male BALB / c mice (4 weeks old, 15 g) were purchased from Coatec Co., BALB / c mice were divided into four groups: normal control group, virus control group, gold extract group after virus infection, and oroxylin A group after virus infection. CVB3 was infected into the abdominal cavity to _{^{1 × 10 6 TCID 50/100}} μL. The golden methanol extract was orally administered to 0.5% CMC (carboxymethyl cellulose) at a concentration of 30 mg / kg, and oroxylin A was dissolved in PBS at a concentration of 10 mg / kg. The drug administration was administered at the same time every day for 5 days, and the body weight was also measured daily during the experiment. After the end of the experiment, the tissue damage (H & E staining) using the pancreas tissue of the mouse was confirmed and the degree of tissue damage including the Acinar cell caused by the virus was confirmed.

4-2. In vivo  Through experiments On CVB3  The golden methanol extract and oroxylin  Antiviral activity of A

After confirming antiviral activity of Norwogonin, oroxylin A, and mosloflavone against EV71, CVB3, and CVA16 through cell experiments, CVB3-infected BALB / c mice were used to examine the antiviral activity against CVB3 The antioxidant activities of golden methanol extract and oroxylin A were measured. Currently coxsackievirus B is the only type of coxsackievirus A, B, and enterovirus in enterovirus that causes infection in mice. Therefore, we conducted a mouse experiment using CVB3 to examine whether gold extracts and Oroxylin A, which exhibit broad-spectrum antiviral activity against Enteroviruses such as EV71, CVB3, and CVA16 in vitro, are actually active in vivo.

As a result, body weight was increased normally in the normal control group, but the viral infection group was decreased by about 10% as compared with the normal control group. The methanol extract group and the oroxylin A treated group showed a decrease of about 10% in body weight and no significant difference from the virus-infected control group as compared with the normal control group (FIG. 9).

We analyzed the tissue damage of the pancreas, which is most known to be the most prominent by CVB3 infection, in order to confirm the improvement effect of tissue damage, even though there is no significant recovery effect on body weight. The pancreas of the pancreas was used for tissue staining. The acinar cells of the pancreas were normal in the normal control group, but most of the acinar cells were damaged by the viral infection in the viral infection control group. However, in the group treated with golden methanol extract and oroxylin A, acinal cells were less damaged than the virus-infected control group (FIG. 10).

As a result of this study, antiviral activity of CVB3 was observed in gold extract and gold chloroform fraction, and antiviral activity of norwogonin, oroxylin A and mosloflavone isolated from golden chloroform fractions were confirmed for EV71, CVB3 and CVA16. Especially in the case of oroxylin A and gold extracts, CBV3-infected mice showed improvement of pancreatic tissue damage. Therefore, norwogonin, oroxylin A and mosloflavone are expected to be used as novel antiviral candidates for enteroviruses.

Experimental Example 5. Toxicity Test

Acute toxicity was tested in ICR mice (male, 6 weeks, purchased from Aruet) according to KFDA specifications. As a result, the gold extract did not show acute toxicity (the mouse did not die) until oral administration of 300 mg / kg.

As a result of the above experiment, it was confirmed that the gold extract can be safely used for the infection of influenza virus.

The extract or compound of the present invention can be administered in the form of the following formulation, and the following formulation examples are illustrative of the present invention, and the contents of the present invention are not limited thereto.

Preparation Example 1. Preparation of powder

SBM extract --------------------------------------- 20 mg

Lactose -------------------------------------------- 100 mg

Talc --------------------------------------------- 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation Example 2. Preparation of tablets

SBB extract --------------------------------------- 10 mg

Corn starch -------------------------------------- 100 mg

Lactose -------------------------------------------- 100 mg

Magnesium stearate ------------------------------- 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation Example 3. Preparation of capsules

norwogonin compound -------------------------------- 10 mg

Crystalline cellulose --------------------------------- 3 mg

Lactose --------------------------------------- 14.8 mg

Magnesium stearate 0.2 mg < RTI ID = 0.0 >

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation Example 4. Preparation of injection

SBC extract --------------------------------------- 10 mg

Mannitol ------------------------------------------ 180 mg

Sterile sterile distilled water for injection ----------------------------- 2974 mg

Na ₂ HPO ₄ 12H ₂ O -------------------------------------- 26 mg

(2 ml) per 1 ampoule according to the usual injection preparation method.

Formulation Example 5. Preparation of a liquid preparation

oroxyline -------------------------------------- 10 mg

Ising Party ----------------------------------------- 10 g

Mannitol -------------------------------------------- 5 g

Purified water -------------------------------------------

Each component was added to purified water in accordance with the conventional liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed and then purified water was added thereto to adjust the whole volume to 100 ml. The solution was filled in a brown bottle and sterilized, .

Formulation Example 6. Preparation of Healthy Foods

SBW Extract ----------------------------------- 1000 mg

Vitamin mixture -----------------------------------

Vitamin A Acetate ---------------------------- 70 g

Vitamin E -------------------------------------- 1.0 mg

Vitamin B1 ------------------------------------ 0.13 mg

Vitamin B2 ------------------------------------ 0.15 mg

Vitamin B6 ------------------------------------- 0.5 mg

Vitamin B12 ------------------------------------ 0.2 g

Vitamin C --------------------------------------- 10 mg

Biotin ----------------------------------------- 10 μg

Nicotinic acid amide 1.7 mg

Folic acid ------------------------------------------- 50 μg

Calcium pantothenate --------------------------------- 0.5 mg

Inorganic mixture -----------------------------------

Ferrous sulfate 1.75 mg < RTI ID = 0.0 >

Zinc oxide - 0.82 mg

Magnesium carbonate --------------------------------- 25.3 mg

Potassium phosphate monohydrate 15 mg

Secondary calcium 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 composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation Example 7. Preparation of health drink

mosloflavone compound 1000 mg

Citric acid --------------------------------------- 1000 mg

Oligosaccharides --------------------------------------- 100 g

Plum concentrate --------------------------------------- 2 g

Taurine ------------------------------------------- 1 g

Add purified water - 900 ml total

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 2-liter container, which was sealed and sterilized, ≪ / RTI >

Claims (4)

Coxsackievirus CVB3 containing the mosloflavone compound as an active ingredient, or an enteroterivirus against the enterovirus of the EV71 serotype of human enterovirus. The present invention relates to a pharmaceutical composition for treating or preventing a disease or condition selected from the group consisting of a skin cancer, a skin rash, a sterile meningitis, herpes zoster, testis, myocarditis, encephalitis, Guillian-Barre syndrome, For the prophylaxis and treatment of infectious diseases caused by enterovirus selected from transverse myelitis, limb paralysis, diabetes, epidemic hemorrhagic keratoconjunctivitis, aseptic meningitis, ulcers, rashes, acute relief paralysis, pericarditis, type 1 diabetes, or pancreatic infections Composition. The present invention relates to a pharmaceutical composition for treating or preventing a disease or condition selected from the group consisting of a skin cancer, a skin rash, a sterile meningitis, herpes zoster, testis, myocarditis, encephalitis, Guillian-Barre syndrome, For the prevention and amelioration of infectious diseases caused by enterovirus selected from transverse myelitis, diarrhea, epidemic hemorrhagic keratoconjunctivitis, aseptic meningitis, ulcer, rash, acute relief paralysis, pericarditis, type 1 diabetes, or pancreatic infection Functional foods. The method of claim 3, wherein
The health functional food is a health functional food in the form of a food, a powder, a granule, a tablet, a capsule, a syrup, a beverage, a gum, a tea, a vitamin complex, or a health functional food.

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