WO2011043631A2 - Pharmaceutical composition for preventing and treating cold, containing reynoutria elliptica extract, fraction thereof or stilbene-based compound - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for relieving cold symptoms or preventing or treating cold, containing a Reynoutria elliptica extract, a fraction thereof or a stilbene-based compound derived from the same as an active ingredient. In addition, the present invention relates to a skin preparation for external use, food and sanitary products containing the composition.

Description

Pharmaceutical composition for the prevention and treatment of colds comprising extracts of kojang-gun, fractions thereof or stilbene compounds

The present invention relates to a composition for improving cold symptoms or for preventing or treating cold symptoms using as an active ingredient, e.g., root extract or fractions thereof. The present invention also relates to external skin preparations, foodstuffs and personal care products containing the composition.

Common cold is a generic term for acute inflammatory diseases of the respiratory mucosa, such as the nose, throat, and bronchus, and includes acute rhinitis, acute pharyngitis, acute laryngitis, acute tonsillitis, bronchitis, and other viral upper respiratory tract infections. . The causes of the common cold are caused by various kinds of viruses, bacteria, irritation of cold and dust, imbalance of body temperature distribution and allergens, but only one of them is caused by cold and dust. It is known that the virus can be caused by a combination of stimulation and imbalance of body temperature distribution, causing a cold. There are about 50 kinds of viruses that cause colds, but the main ones are influenza virus, adenovirus, respiratory syncytial virus, rhinovirus, coxsackievirus, etc. to be. The diseases caused by these viruses have a common common cold syndrome such as runny nose, sneezing, coughing, fever or sore throat, but they are collectively referred to as 'cold', but recently, the cause and symptoms are obvious with the development of medicine. In addition, there is a tendency to call an independent disease, such as allergic rhinitis, and the current cold means a cold in a narrow sense, that is, a viral cold other than influenza.

Colds and influenza are distinguished, especially in complications, with rhinoviruses for colds and influenza viruses for flus. Influenza viruses that cause pandemic influenza are classified into three types, A, B, and C, depending on the antigen type, of which A is the most common antigen mutation and accounts for 90% of the worldwide pandemic virus. . These flu viruses occur worldwide every year and cause respiratory illnesses. They cause severe and painful symptoms such as chills, myalgia, lethargy, respiratory pain, headache, and abdominal pain caused by high fever. If mortality is high for children and the elderly, and complications involving bronchial diseases such as pneumonia and cardiopulmonary disease, the mortality rate is higher.

On the other hand, rhinoviruses account for 30-35% of the causes of adult colds and have more than 100 antigenic types. Infected rhinoviruses do not enter the body and survive by attaching to epithelial cells of the upper airway for 4 to 2 weeks. Unlike the flu virus, which causes pneumonia and kills infected people, rhinoviruses are known to cause runny nose, chills, headaches, and malaise, but they do not harm the body. In other words, living together while living symbiosis is easy to multiply and is known to cause acute respiratory diseases such as asthma, chronic bronchitis. Renovirus is active in early autumn, spring and summer. Investigations into polymerase chain reaction (PCR) studies have shown that the actual range of rhinovirus infections involved in common cold syndrome is at least twice as high as those observed through conventional cell culture techniques (Johnston, Journal of clinical microbiology, 111-117, 1993). This indicates that up to 70-75% of all patients with common cold have ongoing rhinovirus infection as a single infection or as a co-infection.

Coxsackievirus is a common infection in summer or autumn, and is known to cause viral colds along with rhinoviruses. The virus was first discovered in Coxsackie, USA. It is transmitted through contaminated air, water, or feces or saliva from carriers. It is a virus that causes abnormalities in the skin, nerves, circulatory system, and respiratory system.

There is no effective treatment for common cold patients at this time. Some provided therapies have worsened the common cold, such as the administration of aspirin and acetaminophen, which have been reported to have a detrimental effect on the treatment of common cold and can neutralize antibodies and increase nasal organ problems (Graham, J. Infect. Dis, 162: 1277-1282, 1990). Oral alpha-agonists can alleviate hyperemia in many individuals and antihistamines may help, but no actual treatment has been investigated. Prevention or treatment with artificial soluble receptors has not been as successful as expected (Hayden, Antiviral Res., 9: 233-237, 1988). Several attempts to treat patients with common cold with interferon were also negative (Monto, Antimicrobial agents and chemotherapy 33 (3): 387-930, 1989; Sperber, J. Infectious Dis. 160: 700-705, 1989). Recently, Plecarnil®, which inhibits the binding of rhinoviruses through the site of attachment, has also been reported to be negative. Therefore, the development of an effective cold treatment agent is urgently needed.

Meanwhile, Polygonum Cuspidatum (Japanese Knotweed ) is a perennial plant of the genus Madaceae, which grows in Sanya in Korea, and is distributed in Korea, Japan, Taiwan, and China. Ho Jang-geun has been used as a diuretic, pain economy, and sedative in the private sector, and in traditional medicine in Korea and other Asian regions Has been used.

The present inventors, while searching for a composition for the prevention and treatment of colds in natural products, resveratrol isolated from the extract or fractions thereof, or resveratrol isolated from it exhibits antiviral activity against linovirus or coxsackievirus, thereby preventing and treating colds. The present invention has been completed by identifying that it can be used as a composition.

It is an object of the present invention to provide a composition for improving or preventing or treating the symptoms of a cold, comprising an extract of E. coli muscle or a fraction thereof as an active ingredient.

Another object of the present invention is to provide a cold treatment method comprising administering to a subject a composition comprising Escherichia coli extract or a fraction thereof as an active ingredient.

Another object of the present invention is to provide an external preparation for skin, foodstuffs and personal care products containing the composition.

The extracts of the present invention, the fractions thereof, and the stilbene compounds isolated therefrom exhibit excellent effects on the improvement of cold symptoms or prevention and treatment of cold symptoms caused by viruses such as rhinovirus or coxsackie virus, and thus, external skin preparations, foodstuffs It can be widely applied to cosmetics, cosmetics and personal hygiene products.

1 is a diagram showing a resveratrol standard quantity curve.

Figure 2 is a diagram showing chromatogram and MS spectrum of Escherichia coli ethanol extract.

Figure 3 is a diagram showing the chromatogram and MS spectrum of E. coli ethyl acetate fraction.

FIG. 4 is a diagram showing chromatogram and MS spectrum of chloroform: methanol = 20: 1 fraction in Escherichia coli ethyl acetate fraction column chromatography.

Figure 5 is a diagram showing the chromatogram after treatment with E. coli ethanol extract and 12 hours acid.

6 is a diagram showing the inhibitory activity of renovirus (HRV-2, 3, 14) of resveratrol.

7 is a diagram showing renovirus (HRV2) inhibitory activity by mixing resveratrol with a virus and inoculating cells after 1 hour of reaction (before 1h) and inoculating the cells first and then adding resveratrol after 1 hour (after 1h). to be.

FIG. 8 shows micrographs of no vaccinated WI-38 cell lines, micrographs inoculated with each of HRV-2, HRV-3, and HRV-14, and micrographs of 100 μM of each virus and resveratrol. Is a diagram showing.

9 is a diagram showing the inhibitory effect of cossackievirus (CVA21) proliferation of resveratrol.

10 to 12 are diagrams showing the inhibitory effect of inflammatory factor expression by cossackievirus (CVA21) induction of resveratrol. 10 is TNF-α, FIG. 11 is IL-6, and FIG. 12 is a diagram showing the expression inhibitory effect of MCP-1.

In one embodiment, the present invention relates to a composition for improving or preventing or treating cold symptoms, including cold root extract or fractions thereof as an active ingredient.

In the present invention, the E. coli extract is extracted with a solvent selected from the group consisting of water, a lower alcohol having 1 to 4 carbon atoms and a mixed solvent thereof, preferably methanol, ethanol or butanol. In addition, in this invention, the said extract shall contain the extract obtained by an extraction process, the dilution or concentrate of an extract, the dried material obtained by drying an extract, or any of these modifiers or refined products.

In the present invention, a method for obtaining Hojanggeun extract is as follows. The low-strength alcohols of C ₁ -C 4 to C ₄ , such as water, methanol, ethanol and butanol, in volume of about 2 to 20 times, preferably about 3 to 5 times, dry weight Using a polar solvent or a mixed solvent having a mixing ratio of about 1: 0.1 to 1:10 as the elution solvent, the extraction temperature is 20 to 100 ℃, preferably at room temperature, the extraction period is about 12 hours to 10 days Preferably, the extraction is performed using extraction methods such as hot water extraction, cold needle extraction, reflux cooling extraction, or ultrasonic extraction for 3 days to 7 days. Preferably, the extract is filtered 1 to 5 times by cold extraction and subjected to filtration under reduced pressure, and the filtrate is concentrated under reduced pressure at 20 to 100 ° C., preferably at room temperature using a vacuum rotary condenser, and soluble in water, lower alcohols, or a mixed solvent thereof. One colic root extract can be obtained.

The extract of K. koji with the prophylactic and therapeutic activity of the common cold can be extracted from various organs of natural, hybrid, and mutant plants, for example roots, stems, leaves, flowers, trunks of berries, peels of berries, as well as plant tissues. Extractable from the culture and most preferably from the roots of the roots

In the present invention, the solvent fractions of the extracts of the roots of the extract may be obtained by suspending the extract of the roots of the extract with a nonpolar solvent such as n -hexane, ethyl acetate or chloroform, respectively, to obtain a polar solvent fraction and a nonpolar solvent fraction. In the present invention, as a specific aspect, the solvent fraction is provided with water fraction, n -hexane fraction, ethyl acetate fraction or chloroform fraction.

In the present invention, a method for obtaining a fraction of the extract of Keunjangeun is as follows. Suspension of the crude extract obtained above is suspended in distilled water, and then 1 to 10 times by adding a nonpolar solvent such as hexane, ethyl acetate or chloroform in a volume of about 1 to 100 times, preferably about 1 to 5 times, the suspension. Preferably it can be obtained by extracting and separating the nonpolar solvent soluble layer in two to five times. It is also possible to further carry out conventional fractionation processes (Harborne JB Phytochemical methods: A guide to modern techniques of plant analysis, 3rd Ed. P6-7, 1998). Specifically, after suspending the crude root extract in water, an equivalent amount of n-hexane and chloroform can be obtained by continuous extraction using a solvent to obtain a soluble extract of each solvent of the root extract, and more specifically, the crude extract of After suspending in water, the same amount of n-hexane was mixed and fractioned to obtain n-hexane soluble fraction and water soluble fraction, and chloroform was added to the water soluble fraction to obtain chloroform soluble fraction and water soluble fraction. can do.

The present invention is characterized in that the extract of Kojang-geun and its fractions contain resveratrol, a stilbene compound represented by the following formula (1).

Formula 1

Ethanol extracts and fractions of E. coli of the present invention showed high antiviral efficacy at low concentrations against various strains of rhinovirus (HRV-2, HRV-3) (Table 3).

In addition, extracts and fractions of K. koji muscle showed high content of resveratrol, respectively, and resveratrol showed high levels of antilinovirus and anticoxakivirus activity (FIGS. 6 to 12), and thus, containing resveratrol. Knotweed extract and fractions can also be seen to be suitable for the prevention and treatment of colds.

As another aspect, the present invention relates to a composition for improving or preventing or treating cold symptoms, including resveratrol represented by Formula 1 as an active ingredient.

When the composition of the present invention includes resveratrol as an active ingredient, resveratrol may be used in the form of a pharmaceutically acceptable salt, and includes all salts, hydrates, and solvates prepared by conventional methods.

As salts are acid addition salts formed with pharmaceutically acceptable free acids. Acid addition salts are prepared by conventional methods, for example by dissolving a compound in an excess of aqueous acid solution and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Equivalent molar amounts of the compound and acid or alcohol (eg, glycol monomethyl ether) in water can be heated and the mixture can then be evaporated to dryness or the precipitated salts can be suction filtered. In this case, organic acids and inorganic acids may be used as the free acid, and hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, and the like may be used as the inorganic acid, and methanesulfonic acid, p -toluenesulfonic acid, acetic acid, trifluoroacetic acid, and maleic acid may be used as the organic acid. (maleic acid), succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid (gluconic acid), galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanic acid, hydroiodic acid, and the like can be used. It is not limited.

Bases can also be used to make pharmaceutically acceptable metal salts. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and then evaporating and drying the filtrate. In this case, as the metal salt, it is particularly suitable to prepare sodium, potassium or calcium salt, but is not limited thereto. Corresponding silver salts may also be obtained by reacting alkali or alkaline earth metal salts with a suitable silver salt (eg, silver nitrate).

Pharmaceutically acceptable salts of resveratrol include salts of acidic or basic groups which may be present in the compound of resveratrol, unless otherwise indicated. For example, pharmaceutically acceptable salts may include sodium, calcium and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, Dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p -toluenesulfonate (tosylate) salts, and the like. It can be prepared through.

As used herein, the term "amelioration of cold symptoms or prevention or treatment of a cold" includes the prevention and complete or partial treatment of a cold. It also includes reducing cold symptoms, ameliorating cold symptoms, alleviating the pain of a cold or its symptoms, reducing the incidence of colds, or any other change in a patient that increases treatment outcome.

Symptoms of a cold in which the composition of the present invention is effective are sneezing, runny nose, nasal obstruction, nasal congestion, laryngitis or sore throat, cough, sore throat, asthma and headache, fever, chills and poor condition It includes one or more of the common symptoms, such as ones that do not. Therefore, the present invention is a cough, sneezing, runny nose, myalgia, sore throat, nasal obstruction, laryngitis, sore throat, hoarseness, asthma, headache, sinus pain, rhinitis, mucosal boil, pharyngitis, bronchitis chills, chills and nausea It is effective in one or more amelioration, treatment or prevention.

Resveratrol of the present invention is suitable for the prevention and treatment of colds by rhinovirus, a cold virus.

In a specific embodiment of the present invention, the inhibitory effect of resveratrol on rhinovirus was measured. When resveratrol was mixed with a virus and inoculated into cells after 1 hour of reaction, the virus was first inoculated into cells and after 1 hour, resveratrol was added. In the case of (after 1h), renovirus (HRV2) inhibitory activity was excellent in all cases, and thus resveratrol directly acts on the renovirus, indicating that the antiviral effect and the viral infection prevention effect are also shown (FIGS. 6 to 8). .

Resveratrol of the present invention is suitable for the prevention and treatment of colds by the coxsackie virus, a cold virus.

In a specific example of the present invention, resveratrol was determined to inhibit the proliferation of coxsackieviruses. It was confirmed that resveratrol inhibited the proliferation of cossackie virus at significantly low concentrations (Table 4 and FIG. 9). Resveratrol was found to significantly inhibit TNF-α, IL-6 and MCP-1, which are inflammatory factors induced by coxsackievirus (Figs. 10 to 12), and resveratrol acts directly on the coxsackievirus to prevent antiviral Efficacy and virus infection prevention effects were also confirmed.

The composition for improving or preventing or treating cold symptoms of the present invention can be used in a variety of products including foodstuffs for humans and animals, pharmaceutical products, veterinary products, cosmetics, personal care products and household products.

Specifically, the composition of the present invention can be used as an external preparation for skin, and preferably used as an external preparation for skin in the form of an ointment, lotion, spray, patch, cream, powder, suspension, gel or gel.

In addition, the compositions of the present invention can be used in foodstuffs, preferably margarine, fat continuous or water continuous or bicontinuous spreads, fat reduced spreads, chocolate, or chocolate coating Or confectionery such as chocolate fillings or bakery, confectionery, gum, ice cream, ice cream coatings, ice cream ingredients, dressings, mayonnaise, cheese, cream substitutes, dry soups, drinks, cereal bars, sauces, snack bars, dairy products, clinical It can be used as a nutritional or pediatric preparation.

In addition, the composition of the present invention can be used as personal hygiene products, preferably soap, cosmetics, wet wipes, tissue paper, shampoo, mouthwash, skin cream, face cream, toothpaste, lipstick, perfume, make-up, foundation, It can be used as a ball touch, mascara, eye shadow, sunscreen lotion, hair care product, air freshener or cleansing gel.

E. coli extract of the present invention or a fraction thereof, or resveratrol is derived from e.g. root, a drug that has been used for a long time as a herbal, there is no problem such as toxicity and side effects.

The pharmaceutical composition for the prevention and treatment of colds of the present invention may comprise 0.0001 to 10% by weight, preferably 0.001 to 1% by weight of the extract and its fractions or compound 1 based on the total weight of the composition.

The pharmaceutical composition comprising E. coli extract, fractions thereof, or stilbene compound of the present invention may further include appropriate carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

E. coli extracts, fractions or stilbene compounds of the present invention may be used alone or in combination with other pharmaceutically active extracts, fractions or compounds as well as in a suitable collection.

Composition comprising the E. coli extract of the present invention, fractions or stilbene-based compounds can be treated for the cold, including administering to the subject.

The composition comprising the extract of E. coli according to the present invention, its fractions or stilbene compounds, oral formulations, external preparations, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. It can be formulated in the form of suppositories and sterile injectable solutions. In the present invention, carriers, excipients, and diluents that may be included in the composition comprising the extract of K. koji, its fractions or stilbene-based compounds include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, and starch. , Acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil Can be mentioned. When formulated, 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 such solid preparations include at least one excipient such as starch, in the renal vera extract, fractions thereof, or resveratrol separated therefrom. Calcium carbonate, sucrose or sucrose, lactose, gelatin and the like are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid preparations for oral use may include various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to water and liquid paraffin, which are commonly used to include suspensions, solutions, emulsions, and syrups. 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 oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of E. coli extract, fractions thereof, or resveratrol of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration, and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, it is preferable to administer Escherichia coli extract or fractions of the present invention at 0.0001 to 100 mg / kg per day, preferably at 0.001 to 100 mg / kg, and resveratrol at 0.0001 to 10 mg / kg per day. As an example, it is preferable to administer at 0.001 to 10mg / kg. Administration may be administered once a day or may be divided several times.

The pharmaceutical composition of the present invention can be administered to various mammals such as rats, mice, livestock, humans, and the like. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

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

Example 1. Preparation of Keun-Geun Extract and Fraction

Ho Jang Keun was washed with water, dried in the shade, and then powdered with Waring brand. 3.6 kg of powdered rye roots were added to 20 l of ethanol, followed by cold extraction at room temperature for 7 days, followed by filtration under reduced pressure with a filter paper (Watman, USA), and the filtrate extract was extracted after removing the ethanol solvent at room temperature with a vacuum rotary concentrator. As a residue, 146 g of KJ extract was obtained.

The crude extract was suspended in 1 L of water and mixed with the same amount of ethyl acetate. The crude extract was repeated four times to obtain 4 L of ethyl acetate fraction. The ethyl acetate soluble fraction was concentrated under reduced pressure to obtain 80 g of an ethyl acetate soluble extract.

40 g of the ethyl acetate soluble extract obtained above was subjected to silica gel column chromatography using a step gradient solvent system consisting of chloroform: methanol = (100/0 to 1/1) to give chloroform containing resveratrol. Methanol = 20: 1 fraction 830 mg was obtained.

The chloroform: methanol = 20: 1 fraction in the E. coli ethanol extract, ethyl acetate fraction, and ethyl acetate fraction column chromatography obtained above was analyzed using LC-MS to determine the resveratrol content.

Example 2. Analysis of Resveratrol Content of Kwon-Geun Extract and Fraction

2-1. Resveratrol  Standard Quantitative Curve

In the analysis conditions of the standard, HPLC was used Agilent Technologies 1200 series, analytical column was ZORBAX SB-C18 (Agilent, 5 μm, 4.6x150 mm), 60% methanol as the analytical solvent. The analysis was performed at 210 nm, and the flow rate was 0.2 mℓ / min, and the sample was analyzed after injection of 2 μℓ. Resveratrol standard compounds were prepared at concentrations of 1.25 mg / ml, 0.63 mg / ml, 0.31 mg / ml, 0.16 mg / ml, 0.08 mg / ml, and 0.04 mg / ml, and analyzed by HPLC. The peak of the resveratrol standard in the analyzed chromatogram was observed in 11 minutes, and the standard quantitative curve and the linear equation were obtained from the area of the analyzed peak and the amount of injected resveratrol (Fig. 1).

2-2. Resveratrol Content Analysis of Ethanol Extracts

As analytical conditions for E. coli ethanol extract, HPLC of Agilent Technologies 1200 series was used, and the analytical column was ZORBAX SB-C18 (Agilent, 5 μm, 4.6x150 mm) and 50-90% methanol as an analyte. (Table 1-Solvent analysis conditions). The analysis wavelength was 210nm and the flow rate was 0.2 mℓ / min, and the sample was analyzed after injection of 2 μℓ. In order to confirm the resveratrol content of the E. coli ethanol extract obtained above, the sample was prepared at a concentration of 10 mg / ml and then injected into 2 μl and analyzed using LC-MS. As a result of HPLC chromatography, the peak of resveratrol was observed at 17 minutes, and MS analysis showed a molecular peak of m / z 227 [MH] ⁺ (FIG. 2). As a result of substituting the area of the analyzed peak into the equation of the straight line obtained from the standard quantitative curve, it was found that 1 g of renal ethanol extract contained 12 mg of resveratrol (FIG. 2).

Table 1

2-3. Ho Jang Geun  Ethyl acetate Fraction  And ethyl acetate fraction column  For chloroform: methanol = 20: 1 fraction in chromatography Resveratrol  analysis

The obtained E. coli ethyl acetate fraction was analyzed in the same manner as E. coli ethanol extract. As a result of HPLC chromatography, the peak of resveratrol was observed at 17 minutes, and MS analysis showed a molecular peak of m / z 227 [MH] ⁺ (FIG. 3).

The chloroform: methanol = 20: 1 fraction in the ethyl acetate fraction column chromatography was analyzed in the same manner as the resveratrol standard assay. As a result of HPLC chromatography, the peak of resveratrol was observed in 11 minutes, and MS analysis showed a molecular peak of m / z 227 [MH] ⁺ (FIG. 4).

LC-MS analysis of chloroform: methanol = 20: 1 fractions in ethanol extract and ethyl acetate fraction and ethyl acetate fraction column chromatography showed that resveratrol contained 1.2%, 4.6% and 7.1% in each sample. As a result, it was found that resveratrol contained in 1 kg of Kwonjeok herbal medicine accounted for 2.44 g of 0.24% of dry weight (Table 2-Resveratrol content).

TABLE 2

2-4. After acid treatment Resveratrol  Content change analysis

E. coli ethanol extract (200 mg) was dissolved in 9 ml of methanol and added to 1 ml of 3M sulfuric acid solution into a round bottom flask. The flask was reacted in a 70 ° C. water bath for 12 hours and then terminated with ethyl acetate. In order to analyze the change in the content of resveratrol contained in the ethyl acetate fraction (100 mg), after preparing at 10 mg / ml concentration was injected 2 μl and analyzed using LC-MS. Figure 5 shows that the area of resveratrol-3-O-β-D-glucoside peak (11 minutes) was 41,363 mAU in the ethanol extract HPLC chromatogram, but after 12 hours of acid treatment it was reduced to 26,400 mAU. Could. In addition, it can be seen that the area of the resveratrol peak (17 components) increases from 5,618 mAU to 23,380 mAU (FIG. 5). The resveratrol content of K. ethanol extract was measured as 12 mg / g (ethanol extract) (Table 2), and after 12 hours of acid treatment, the resveratrol content increased about 4 times to 50 mg / g (ethanol extract).

Example 3 Determination of Inhibitory Effect of Rhodiola root Extracts and Fractions on Linoviruses

In order to measure the inhibitory effect of chloroform: methanol = 20: 1 fraction on rhinovirus (HRV-2, 3) in ethanol extract, ethyl acetate fraction and ethyl acetate fraction column chromatography, The following in vitro experiments were performed using WI-38 (ATCC: CCL-75), a human lung fibroblast.

First, WI-38 cells were placed in 96 well microplates at 1 X 10 ⁵ cells / well per well and cultured in medium EMEM (penicillin 100 units, streptomycin 100 μg, 10% FBS). When WI-38 cells became a monolayer, they were washed twice with EMEM medium containing only antibiotics. HRV-2 and HRV-3 strains were diluted to 100 TCID ₅₀ and placed in EP tubes. The final concentration of chloroform: methanol = 20: 1 fraction in ethyl acetate and ethyl acetate fraction and ethyl acetate fraction column chromatography diluted with DMSO (dimethylsulfoxide) was 100 μg / mℓ, 60 μg / mℓ, 30 μg / mL, 10 μg / ml, 1 μg / ml was added to each tube and reacted at 4 ℃ for 1 hour. After 1 hour, pre-washed WI-38 cells were inoculated in 3 wells per concentration, respectively.

Meanwhile, interferon alpha (INF-α), which is known for its antiviral effect, and non-infected, non-administered control, and infected and non-administered control, were inoculated into WI-38 cells after 1 hour of reaction under the same conditions. Incubate in. Infected and non-administered controls were incubated for 3-4 days until complete cytopathic effect (CPE). The cells were photographed by observing the magnification of 50X daily with an inverted microscope. To determine cell viability after 3-4 days of incubation, 10 μl of Cell Counting kit-8 (Dojin, Japan, tetrazolium salt WST-8) was added to each well, followed by reaction at 33 ° C. for 2 hours, and then absorbance at 450 nm. It was. At this time, the rhinovirus inhibitory effect of the compound of the present invention was determined using DMSO-only cells and DMSO, HRV-2, and HRV-3 treated cells as controls.

The antiviral activity of the chloroform: methanol = 20: 1 fraction in ethanol extract, ethyl acetate fraction and ethyl acetate fraction column chromatography of the present invention was determined that the EC ₅₀ value was> 60 μg / ml for HRV-2. , 37.2 μg / ml, 11.5 μg / ml and EC ₅₀ values of HRV-3 strains were 47.8 μg / ml, 44.4 μg / ml, and 11.3 μg / ml (Table 3-CPE reduction effects of HRV). .

From the above results, the chloroform: methanol = 20: 1 fractions in E. coli ethanol extract, ethyl acetate fraction and ethyl acetate fraction column chromatography were good for all strains of rhinovirus (HRV-2, HRV-3). Viral efficacy was confirmed, and it is also judged to have a direct effect on the virus and also show a virus infection prevention effect.

TABLE 3

EXAMPLE  4. Resveratrol  Compound Renovirus  Measurement of inhibitory effects

In order to measure the inhibitory effect of resveratrol against rhinoviruses (HRV-2, 3, 14), the human lung fibroblast WI-38 (ATCC: CCL-75) was used as follows. In vitro experiments were performed.

First, WI-38 cells were placed in 96 well microplates at 1 X 10 ⁵ cells / well per well and cultured in medium EMEM (penicillin 100 units, streptomycin 100 μg, 10% FBS). When WI-38 cells became a monolayer, they were washed twice with EMEM medium containing only antibiotics. HRV-2, HRV-3 and HRV-14 strains were diluted to 100 TCID ₅₀ and placed in EP tubes. Resveratrol diluted with DMSO (dimethylsulfoxide) was added to each tube at a final concentration of 100 μM, 60 μM, 30 μM, 10 μM, 1 μM, and reacted at 4 ° C. for 1 hour. After 1 hour, pre-washed WI-38 cells were inoculated in 3 wells per concentration, respectively.

Meanwhile, interferon alpha (INF-α), which is known for its antiviral effect, and non-infected, non-administered control, and infected and non-administered control, were inoculated into WI-38 cells after 1 hour of reaction under the same conditions. Incubate in. Infected and non-administered controls were incubated for 3-4 days until complete cytopathic effect (CPE). The cells were photographed by observing the magnification of 50X daily with an inverted microscope. To determine cell viability after 3-4 days of incubation, 10 μl of Cell Counting kit-8 (Dojin, Japan, tetrazolium salt WST-8) was added to each well, followed by reaction at 33 ° C. for 2 hours, and then absorbance at 450 nm. It was. At this time, the renovirus inhibitory effect of resveratrol compared with DMSO-only cells and DMSO, HRV-2, HRV-3, HRV-14 treated cells as a control group was determined.

As a result, the interferon alpha (INF-α) used as a positive control showed a low inhibitory capacity of 22% in HRV-2, and 55.6% and 80.8% in HRV-3 and HRV-14, respectively. Resveratrol of the present invention showed high inhibitory activity of 100% and 78% at 60 μM, respectively, in HRV-2 and HRV-3, and 100% inhibitory activity at 90 μM in HRV-14 strains (FIG. 6 ). In addition, as a result of observing the cell morphology on the inverted microscope, virus inoculation alone (HRV-2, 3, 14) showed almost 100% cell denaturation effect due to almost destruction of WI-38 cells, but 100 μM of virus and resveratrol. The treated group confirmed that the WI-38 cells were similar to the control group treated with nothing (FIG. 8).

In addition, in order to perform an experiment comparing resveratrol with pre- and post-infection of HRV-2 strain, the virus inhibitory activity of resveratrol after infection was examined. To this end, WI-38 cell linovirus (HRV-2) washed twice with EMEM medium containing only antibiotics was inoculated and incubated at 33 ° C. for 1 hour. After 1 hour, all the inoculated virus solution was removed, and the final concentration of resveratrol was 100 μM, 60 μM, 30 μM, 10 μM, and 1 μM was added to the medium, and the virus was inoculated into WI-38 cells. Then, the linovirus inhibitory effect represented by the compound of the present invention was determined in the same manner as above.

As a result, when HRV-2 was first reacted with resveratrol for 1 hour and then infected, it showed 100% virus inhibition activity at 60 μM and IC ₅₀ value was 13 μM. In addition, when the virus was first infected and resveratrol was administered 1 hour later, 71.6% of inhibitory activity was observed at the same concentration, and the IC ₅₀ value was 36 μM. (FIG. 7).

From the above results, resveratrol was able to confirm good antiviral efficacy in various strains of rhinovirus (HRV-2, HRV-3, HRV-14) of cold virus, and also acts directly on the virus to prevent viral infection. It is judged to indicate.

Example 5: Inhibition of Coxsackievirus Proliferation by Resveratrol

In order to measure the anti-proliferative effect of resveratrol on coxsackievirus, the following experiment was performed using Hela H-1 cells, which are human cervical cancer cells.

First, inoculated with Coxakivirus A21 (CVA21) of 0.01 MOI (multiplicity of infection) to Hela H-1 cells (1 × 10 ⁵ cells / well) and reacted for 1 hour, and then the supernatant was removed, and the resveratrol compound and 30 EMEM medium containing mM MgCl ₂ was added thereto and incubated at 37 ° C. for 48 hours. As a result, when resveratrol was treated in a concentration-dependent manner, it was confirmed that the proliferation of CVA21 was significantly inhibited and the IC ₅₀ value was 44.7 μM. The growth inhibition effect of pleconaril on CVA21 of the positive control was confirmed that the IC ₅₀ value is 5 nM or less (Table 4 and Figure 9).

Table 4

EXAMPLE  6: Resveratrol  Inhibitory Effect of Coxsackievirus Induction on Inflammatory Factor Expression

In order to measure the inhibitory effect of inflammatory factor expression induced by CVA21 infection of resveratrol, the following experiment was performed using Hela-H1 cells, which are human cervical cancer cells.

To measure the inhibitory effect of inflammatory factor induction by resveratrol CVA21 infection, Hela-H1 cells were dispensed at ⁵ × 10 ⁵ cells / well in 12 well plates and the final concentration of resveratrol was increased from 10 μM to 60 μM 1 The time was treated before. The cells were then inoculated with CVA21 of 0.01 multiplicity of infection (MOI) and infected for 1 hour. After 1 hour, uninfected CVA21 was removed by removing the supernatant, and EMEM medium to which 30 mM MgCl ₂ was added was added and incubated at 37 ° C. for 24 hours. The cultured cells were washed with PBS, washed with PBS, scraped with a scraper and collected in a tube to extract RNA using the RNeasy Mini Elute Cleanup kit. Thereafter, the inhibitory effect of inflammatory factor induction represented by resveratrol was measured in the same manner as above.

As a result, it was confirmed that the expression of inflammatory cytokines TNF-a, IL-6, and MCP-1 significantly increased in the group inoculated with CVA21 compared to the control group not inoculated with CVA21, and pretreatment with resveratrol One group showed a similar tendency to the control group, it was confirmed that significantly inhibit the expression of inflammatory factors induced by CVA21 infection (Figs. 10 to 12). However, the control group pleconaril showed no inhibitory effect on the expression of inflammatory factors induced by CVA21 infection (FIGS. 10-12).

These results suggest that the composition of the present invention has an antiviral effect against linovirus as well as coxsackieviruses, demonstrating that it is effective against colds caused by viral infections.

Claims (15)

1. Composition for improving or preventing the symptoms of a cold, including a common extract of the extract Keun-Geun or a fraction thereof as an active ingredient.
2. The composition of claim 1, wherein the extract is an extract extracted with a solvent selected from the group consisting of water, a lower alcohol having 1 to 4 carbon atoms, and a mixed solvent thereof.
3. The composition of claim 2, wherein the lower alcohol solvent is selected from the group consisting of methanol, ethanol and butanol.
4. The composition of claim 1 wherein the fraction is a water fraction, an ethyl acetate fraction, an n-hexane fraction or a chloroform fraction.
5. The composition of claim 1, wherein the extract of E. coli or its fraction comprises a stilbene-based compound represented by Formula 1 as an active ingredient.

   [Formula 1]

   
6. The composition of claim 1, wherein the cold is an infectious disease caused by rhinovirus.
7. The composition of claim 1, wherein the cold is an infectious disease caused by coxsackievirus.
8. According to claim 1, wherein the composition is cough, sneeze, runny nose, myalgia, sore throat, nasal obstruction, laryngitis, sore throat, hoarseness, asthma, headache, sinus pain, rhinitis, mucosal boils, pharyngitis, bronchitis chills A composition for improving a cold symptom selected from the group consisting of chills and malaise.
9. An external preparation for skin comprising the composition of claim 1.
10. The skin external preparation according to claim 9, which is in the form of an ointment, a lotion, a spray, a patch, a cream, a powder, a suspension, a gel or a gel.
11. A composition for food comprising the composition of claim 1.
12. 12. The composition of claim 11 comprising margarine, fat continuous or water continuous or bicontinuous spreads, fat reduced spreads, chocolate, chocolate coating or chocolate fillings or bakery loaf, confectionary, Food composition selected from the group consisting of gum, ice cream, ice cream coatings, ice cream contents, dressings, mayonnaise, cheese, cream substitutes, dried soups, drinks, cereal bars, sauces, snack bars, dairy products, clinical nutrition and pediatric preparations .
13. A personal hygiene product comprising the composition of claim 1.
14. The method of claim 13, wherein the soap, cosmetics, wet wipes, tissue paper, shampoo, mouthwash, skin cream, face cream, toothpaste, lipstick, perfume, make-up, foundation, ball touch, mascara, eyeshadow, sunscreen lotion, hair A personal care product selected from the group consisting of a care product, an air freshener and a cleaning gel.
15. A method of treating colds, comprising administering the composition of claim 1 to a subject.

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