KR100950445B1 - Antiviral agents with inhibitory activities on on avian and swine influenza virus or novel influenza virus by compounds isolated from glycyrrhiza uralensis - Google Patents

Abstract

PURPOSE: A Glycyrrhiza uralensis extract and calcone based compounds suppressing neuraminidase are provided to treat novel swine-origin influenza. CONSTITUTION: A Glycyrrhiza uralensis extract for preventing or treating avian and swine ingluenza contains is extracted using ethanol or methanol and contains chalcone based compound. The chalcone based compound is Licochacone F, Echicnatin, Licochalcon D, Licochalcone A, Isoliquiritigenin, Licoagrochalcone A, 5-Prenylbutein and Kanzonol C. A functional health food for preventing or treating diseases relating to avian and swine influenza contains Glycyrrhiza uralensis extract. A pharmaceutical composition for preventing or treating diseases relating to avian and swine influenza contains Glycyrrhiza uralensis extract or chalcone compouns as active ingredient. The pharmaceutical composition also contains pharmaceutically acceptable carrier or excipient.

Description

Antiviral agents with inhibitory activities on on Avian and Swine influenza virus or Novel influenza virus by compounds isolated from Glycyrrhiza uralensis}

The present invention is licorice ( Glycyrrhiza The present invention relates to a compound which inhibits neuraminidase derived from uralensis ) and a composition for use in the prevention and treatment of avian and swine influenza and swine flu comprising the same as an active ingredient.

Chalcone and more particularly, that after extraction Concentration of the licorice with ethanol or methanol saturated in solution and represented by the following general formula (1) obtained by separation and purification of pure by chromatography from the fraction or combinations of ethyl acetate fractions were fractions with ethyl acetate (chalcone) The present invention relates to a neuraminidase inhibitor compound comprising any one of a series of compounds and a composition for preventing and treating a virus neuraminidase inhibitory related disease such as a cold, avian and swine influenza, swine flu, etc. containing the same as an active ingredient.

Influenza viruses are orthomyxoviruses belonging to the orthomyxo family and are classified into A, B and C types. Type A is a virus that has been confirmed mainly in humans and has been confirmed in pigs, other mammals, and various wild birds compared to types B and C. Recently, Avian influenza, swine influenza and Novel flu, which are a problem worldwide, belong to the type A virus.

There are two proteins on the surface of the influenza virus: hemagglutinin (HA) and neuraminidase (NA) .There are 16 types of hemagglutinin and 9 types of neuraminidase, so 15 × 9 = 144 types of influenza viruses can occur. Influenza infections in birds are mainly related to H5, H7 and H9 types, with only three types of hemagglutinin (H1, H2, H3) and two types of neuraminidase (N1 and N2) in humans. As a rule, humans are not infected with avian influenza. Recently, however, avian influenza viruses have been transmitted directly to humans without undergoing genetic recombination.

Avian influenza spreads quickly through wild birds and migratory birds that come to Korea, in addition to contacting poultry such as chickens, ducks and turkeys. Avian influenza virus is periodically introduced and spread from Southeast Asia, where avian influenza has occurred, and can be transmitted from China through spring yellow dust. Avian influenza, a global viral disease of animals, has been incurred since 1996 in Korea, killing 5.3 million chickens and ducks, causing more than 150 billion won in direct damage. More than 10 livestock are slaughtered, causing huge economic losses for the poultry industry.

The swine influenza virus was reported for the first time in 1918. Usually, swine has a human influenza receptor (N-acetylneuraminic acid α2,6 galactose) and avian influenza receptor (N-acetylneuraminic acid α2,3). It is known to have a galactose, which acts as a virus mixing vessel for the avian influenza virus to infect humans through reassortment or adaptation in pigs.

The Novel flu, which was first introduced in Mexico in 2009 and is spreading around the world, is thought to be a disease that can be transmitted to humans by the swine flu virus H1N1. Swine flu is basically like a cold, high fever, cough, etc. There is a possibility that the virus becomes more lethal than humans. The number of people infected with the H1N1 flu recorded by the World Health Organization (WHO) reached 34,200 by the beginning of October 2009, and the death toll exceeded 4,000. Upgraded to a pandemic phase. Common symptoms of swine flu are very difficult to distinguish from the common flu. Swine flu shows symptoms of fever, sore throat, cough, runny nose or stuffy nose, similar to the common flu, and recent reports related to swine flu show more severe fatal lung damage than the common flu. Treatment is a very important problem. Therefore, it is necessary to secure a vaccine to prevent the H1N1 flu, and it is necessary to produce the vaccine and inoculate nationally before the H1N1 flu spread rapidly. However, it is almost impossible to make a vaccine that prevents all RNA viruses that produce antigenic variation every time as a preventive national vaccine. Therefore, in order to prepare for the development of avian and swine influenza, swine flu or a new pathogen mixed with the worst possible avian influenza and swine flu, it is necessary to develop a therapeutic agent that can prevent and treat it.

Currently, the only treatments for avian and swine influenza and swine flu are oralamivir phosphate, oral inhaled zanamivir, which is an oral therapeutic agent developed as a selective inhibitor of neuraminidase of viral origin. Side effects of Tamiflu, which is widely used as an oral treatment, have been reported such as nausea, vomiting, nervous or psychological problems, and in Japan, there are 15 cases of death in pediatric patients confirmed after Tamiflu approval. In addition, it should be prepared for the development of resistant virus against Tamiflu, which is used as an oral treatment. Recently, the emergence of Tamiflu-resistant virus, which is fatal to the spread of swine flu, has been reported, and even human-to-human transmission of Tamiflu-resistant virus has been found. Therefore, the development of virus treatments against avian and swine influenza and swine flu is essential for human health and safety, especially in the war against the pandemic avian and swine flu and swine flu.

In the process of infection and proliferation of the virus, the virus breaks down sialic acid, which is present outside the cell, using neuraminidase made by the virus to make a new virus inside the cell and spread to other cells after infection. It is necessary to do Tamiflu-resistant avian influenza viruses are known to occur through three genetic variations (Arg292Lys, Asn274Ser, His294Tyr) that normally alter the amino acids of Tamiflu's receptor protein to which Tamiflu binds. Of the three genetic variants, the 294 His, the most highly expressed variant of the Tamiflu-resistant avian influenza virus, changes to Tyr when its hydrophobic residues do not bind to Tamiflu receptors. . It is a virus found in the United States and Europe with a 10-25% chance of being found in the 2007-2008 season, increasing the risk of future mutations. The emergence of these Tamiflu-resistant viruses therefore provides a justification for humans to continue developing new viral therapies.

In the present invention, the neuraminidase inhibitors against avian and swine influenza and swine flu targeting the food and medicinal plants that can be adapted immediately if the effect is confirmed in view of the urgency against defense measures of avian and swine influenza and swine flu. Explored. In general, finding new active ingredients from natural medicines used in traditional medicine has many advantages over experimental modification of existing drugs to develop new ingredients of medicine. In particular, since these active ingredients have been used for a long time, there is little concern about toxicity due to drugs. This fact can be confirmed from the development of Tamifludo starting material, which is used as a treatment for swine flu, as the main ingredient of 'Staranis' fruit, an indigenous plant in China.

Licorice that is the subject of the present invention ( Glycyrrhiza uralensis (Licorice) is a perennial herb that is native to eastern Siberia and northeastern China, and is called licorice because of its roots. It is an important herbal medicine that is almost included in herbal medicine. In the case of using licorice extract as a patent and literature related to licorice, licorice is mentioned in "Health food composition for preventing and treating allergic rhinitis and cold and its manufacturing method," (Korean Registered Patent No. 566542). It refers to one of the mixtures and is not related to this patent which identifies the structure by separating the compound showing activity in the extract.

In the case of a patent using a compound known in licorice, a patent using lyocacalcon E similar to compound 4 (lyocacalcon A) claimed in the present invention is "Ricocalcon E and an anticancer composition comprising the same (Korean Patent No. 564811)". , "Calcon derivatives exhibiting antithrombotic effect, pharmaceutical compositions comprising the same, and methods for preparing the same (Korean Patent No. 380981)", and the like, but there are no virus-related patents. Recently, there is a patent for influenza virus using a stilbene-based compound resveratrol, "the use of resveratrol for the manufacture of a medicament useful for the treatment of influenza virus infection (Korean Patent Publication No. 2005-71627)", but the content of the present invention is also different.

The present invention purely isolates the chalcone-based compounds from the organic solvent extracts of licorice and compares these compounds with their activities against avian and swine influenza virus, swine flu and Tamiflu-resistant swine flu virus (His294Tyr). It was confirmed that the compounds of the present invention have inhibitory activity against avian and swine influenza and swine flu.

An object of the present invention is a chalcone family obtained by purely isolated and purified from licorice, which prevents and treats avian and swine influenza virus, swine flu, and tamiflu-resistant swine flu neuraminidase, thereby showing prophylactic and therapeutic effects on avian influenza and swine flu-related diseases. The present invention provides a composition containing each or two or more compounds as an active ingredient.

In view of the above, the present inventors have collected various native plants and herbal medicines and used a culture solution of avian and swine influenza virus or cloned neuraminidase of swine flu and Tamiflu-resistant swine flu virus, and then introduced them into cells. Licorice was selected as a candidate plant by investigating the inhibitory effect on miniday enzyme. Subsequently, extraction with organic solvent alcohol (methanol, ethanol, propanol, etc.) or water-soluble alcohol from licorice, distribution of organic solvent and water, method by column chromatography, well-known methods used for separation extraction of plant components, etc. Or it can obtain easily by combining suitably. The crude extract can be further purified according to a commercial method as needed.

According to the present invention, licochalcone F (compound 1) represented by the following Chemical Formula 1 of a plurality of chalcone-based compounds obtained by extracting licorice with alcohol and separating and purifying pure water using chromatography and confirming the structure through chemical structure and physicochemical characterization ), Echinatin (Compound 2 ), Lycochalcone D (Compound 3 ), Lycochalcone A (Compound 4 ), Isocristygenin (Compound 5 ), Ricoagrochalcone A (Compound 6 ), 5-Phenylbutane ( The present invention was completed after the activity of compound 7 ) and cansonol C (compound 8 ) was confirmed by measuring the inhibitory effect on the virus-derived neuraminidase and Tamiflu-resistant neuraminidase.

The present invention is characterized by including at least one compound of the above compounds as an active ingredient to inhibit neuraminidase to prevent or treat avian and swine influenza, swine flu-related diseases.

The compound is a step of separating and purifying pure chalcone-based compound from the organic solvent extract of licorice; Examining the chemical structure and physicochemical properties of the chalcone-based compound obtained in the step; Making neuraminidase of the swine flu virus, neuraminidase of the Tamiflu resistant swine flu virus (His294Tyr); Activity is measured through the step of measuring the inhibitory action of the avian influenza virus (H9N2) and swine influenza virus (H1N1) of the chalcone-based compound against the neuraminidase and the swine flu virus and the Tamiflu-resistant swine flu virus I knew.

Inhibitory compounds of avian influenza virus (H9N2) and swine influenza virus (H1N1) against neuraminidase and swine flu virus and Tamiflu-resistant swine flu virus neuraminidase from organic liquor from licorice (alcohol, alcohol aqueous solution, Known methods used for the separate extraction of plant components, such as extraction with ethyl acetate, ether, acetone, chloroform, etc., distribution of hexane and water, use of adsorption resins such as DIION HP-20 resin, method by column chromatography It can be obtained easily by combining single or suitably. The crude extract can be further purified according to a commercial method as needed.

The crude extract can be further purified according to a commercial method as needed. The chromatography used in the present invention includes silica gel column chromatography, L-20 column chromatography, thin layer chromatography (TLC) and high performance liquid chromatography (TLC). high performance liquid chromatography) and the like.

The chalcone-based compound according to the present invention can be easily separated from licorice, which is a legume, and also has high stability, and thus can be used as an additive for food and medicine.

Pharmaceutical compositions comprising extracts according to the invention, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used. Examples of carriers, excipients and diluents that can be included in the composition containing the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When 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 the solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, and the like. Mix is prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. 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.

The dosage of the active ingredient will vary depending on the age, sex and weight of the subject to be treated, the particular disease or pathology to be treated, the severity of the disease or pathology, the route of administration and the judgment of the prescriber. Dosage determination based on these factors is within the level of skill in the art and generally dosages range from 0.01 mg / kg / day to approximately 2000 mg / kg / day. More preferred dosage is 0.1 mg / kg / day to 500 mg / kg / day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect. The extract of the present invention can be administered to mammals such as mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections. Since the extract of the present invention has little toxicity and side effects, it is a drug that can be used safely even when taken for long periods of time.

In addition, the present invention provides a dietary supplement for the prevention of algae and swine influenza and swine flu, including the licorice extract and food acceptable additives. The health functional food of the present invention includes the form of tablets, capsules, pills, or liquids, and the food to which the compound of the present invention can be added includes, for example, various foods, beverages, gums, teas, vitamins, and the like. Complex, health functional foods, and the like. In detail, the present invention provides a functional food for the prevention and treatment of swine flu-related diseases, including an extract containing licorice extract as an active ingredient and a food acceptable additive.

The present invention can be used as feed additives and therapeutic substances for the purpose of preventing and treating animal diseases including the licorice extract and the chalcone compound. The feed additive and the therapeutic substance of the present invention can be used for infectious enteritis caused by bacterial infection, which is a common common disease related to neuraminidase, which commonly occurs in animals and humans, and especially for preventing diseases related to swine flu, bird, and swine influenza. And animal feed additives for the treatment, animal drugs are provided.

Licorice extract and chalcone-based compounds according to the present invention have a therapeutic effect of non-competitively inhibiting neuraminidase of avian and swine influenza virus or swine flu virus neuraminidase and Tamiflu-resistant swine flu virus, and Tamiflu resistant to Tamiflu. Resistant H1N1 virus could similarly inhibit neuraminidase.

Moreover, when Tamiflu and the compound of the present invention were treated in combination with avian influenza (H9N2) and swine influenza (H1N1) viruses, the amount of Tamiflu could be reduced (the result is not shown in this patent). It has been confirmed that the neuraminidase of these species and the neuraminidase of the Tamiflu-resistant H1N1 virus have a similar function.

Thus, licorice extract and chalcone-based compounds isolated therefrom inhibit the neuraminidase of avian influenza virus, swine influenza virus, swine flu virus, and Tamiflu-resistant swine flu virus to prevent avian influenza and swine flu-related diseases. The effectiveness of the treatment can be expected.

In addition, since it has been used as a copulation agent to neutralize the medicinal properties of various Chinese medicine ingredients for a long time, it is extracted and purified from licorice which can guarantee safety. It can be very usefully used and applied.

Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided to ensure that the contents introduced herein are thorough and complete, and that the spirit of the present invention to those skilled in the art can fully convey.

< Example  1: Study on Solvent Extraction Conditions of Active Compounds from Licorice>

To compare the degree of solvent extraction of the active substance from licorice, using the solvent of distilled water, 30-100% ethanol aqueous solution, methanol, acetone, ethyl acetate, chloroform to compare the extraction degree of the active ingredient with respect to licorice 100g Ultrasonic extraction was performed three times for 2 hours using 300 ml of solvent, and the extract was concentrated under reduced pressure. As a result of comparing the amount of the extracted extract, it was shown that the amount of extract from 3.6g (acetone) to 32.5g (distilled water). Then, in order to compare the activity of these extracts with respect to neuraminidase, each extract was prepared at the final concentration of 20 μg / ml, and the activity of each extract was determined using the virus culture medium of Example 4-2 and Example 6 Measured using. As can be seen from the results shown in Table 1, the extract was good in 50 ~ 100% ethanol, methanol extract, but 70% ethanol by comparing the amount and activity of the extraction solvent and the amount of solvents that are acceptable to the human body The extract was set to the optimum extraction conditions.

 Condition  Extraction amount  Inhibitory activity against porcine influenza virus-derived neuraminidase (20 μg / mL)  30% ethanol  26.0 g  19.3%  50% ethanol  23.1 g  36.9%  70% ethanol  18.0 g  38.5%  90% ethanol  14.7g  34.5%  ethanol  7.6 g  35.1%  Methanol  11.5 g  33.8%  Acetone  3.6 g  5% less than  Ethyl acetate  5.6 g  12.4%  chloroform  6.5g  5% less than  Distilled water  32.5 g  5% less than

< Example  2: solvent extract from licorice Carlton  Isolation of Compounds>

3 kg of dried licorice root was sonicated three times for 4 hours using 10 L of 70% ethanol. The 70% ethanol extract (hereinafter referred to as 'A fraction') was concentrated under reduced pressure, and then 350 g of the extract was suspended with water (2 L), followed by n-hexane (2 L) extract (hereinafter referred to as 'B fraction'), Solvent fractionation was sequentially performed with ethyl acetate (2 L) extract (hereinafter referred to as the 'C fraction') and butanol (2 L) extract (hereinafter referred to as the 'D fraction'), followed by algae concentration under reduced pressure in each solvent fraction. Influenza virus and swine influenza virus neuraminidase activity was measured, and the ethyl acetate fraction showed strong activity.

50 g of ethyl acetate fractions were subjected to silica gel column chromatography under a solvent gradient of hexane-acetone 4: 1 to 0: 1 to obtain 6 fractions (fr. 1 to 6). Among these fractions, Fraction 2 (10.5 g) showing strong activity was loaded onto Sephadex LH-20 (Sephadex LH-20) resin and eluted with methanol to obtain 6.2 g of Compound 4 (Licocalcon A). Reverse phase (RP-18) column chromatography was performed again under the condition that fraction 3 (4.5 g) was sequentially increased from 5: 5 to 10: 0 using MeOH-H ₂ O mixed solvent. Was obtained (fr.3.1-3.8). Small fraction F3.2 (120 mg) was purified by HPLC [YMC Pak C ₁₈ column 20 × 150 mm using MeOH—H ₂ O (0-55 min: 62% MeOH, 60 min: 100% MeOH); 4 μm particle size 3 ml / min; UV detection: 254 nm] was carried out to obtain Compound 2 (Echinatin, 20 mg, t _R = 25.0 min) and Compound 7 (5-prenylbutane, 1.5 mg, t _R = 50.0 min). Small fraction F3.3 (250mg) with _{MeOH-H 2 O (0-55min:} 65% MeOH, 60min: 100% MeOH) subjected to HPLC in the same conditions to release the small fraction F3.2 using Compound 3 ( Ricokalcon D, 17 mg, t _R = 42.0 min), Compound 1 (Ricochalcon F, 5.0 mg, t _R = 50.0 min) were obtained and 68% solvent of methanol used from small fraction F3.4 (250 mg) was obtained. Compound 5 (Isocristygenin, 18.0 mg, t _R = 43.0 min) and Compound 6 (Ricoagrochalcon A, 2.0 mg, t _R = 48.0 min) were obtained. Finally, a portion of subfraction F3.6 was taken to obtain compound 8 (canzonol C, 4.0 mg, t _R = 60.0 min) under conditions using 86% MeOH-H ₂ O (see FIG. 1).

The neuraminidase activity was confirmed by the method of Example 6, the results are shown in Example 7 and Table 2 and Table 3.

< Example  3: Calcon  Physical and chemical properties and chemical structure analysis of compounds>

In Example 2, the structure of 8 compounds isolated from licorice was analyzed. The chemical structure of the compound was analyzed based on ¹ H and ¹³ C-NMR analysis of molecular weight and nuclear magnetic resonance analysis obtained using an ESI mass spectrometer.

As a result, the structure-determined compound had the structure shown in Formula 1 above, and summarized the chemical properties of the separated compound and the ¹ H, ¹³ C-NMR results of the compound 1 to compound 8 are shown below.

Rico chalcone F (Licochalcone F, Compound 1 ): Yellow needle; UV (MeOH) λ _max nm (log ε): 260 (3.76), 380 (4.09); IR (KBr) Ｖ _max 3420, 2965, 1601, 1557, 1445, 1285, 1166 cm ⁻¹ ; EI MS m / z (rel. Int.): 354 ([M] ⁺ , 27), 349 (10), 324 (23), 323 (100), 137 (42); HREI MS m / z 354.1465 [M] ⁺ ( Calcd . For C ₂₁ H ₂₂ O ₅ , 354.1467); ¹ H-NMR (CD ₃ COCD ₃ , 500 MHz) δ 7.98 (1H, d, J = 15.5 Hz, H-β), 7.64 (1H, d, J = 15.5 Hz, H-α), 7.60 (1H, d, J = 8.0 Hz, H-6 '), 7.58 (1H, s, H-6), 7.56 (1H, d, J = 8.0 Hz, H-5'), 6.94 (1H, d, J = 2.1 Hz, H-3 '), 6.58 (1H, s, H-3), 6.30 (1H, dd, J = 17.0 and 11.0 Hz, H-2 "), 5.04 (1H, dd, J = 17.0 and 1.5 Hz , H-3 "), 4.99 (1H, dd, J = 11.0 and 1.5 Hz, H-3"), 3.87 (3H, s, OCH ₃ ), 1.51 (6H, s, H-4 ", H-5 ¹³ ) C-NMR (CD ₃ COCD ₃ , 125 MHz) δ 188.5 (C = O), 159.9 (C-2 ', s), 159.8 (C-2, s), 150.5 (C-4, s ), 148.7 (C-2 ", d), 145.8 (C-4 ', s), 139.8 (C-β, d), 132.5 (C-1', s), 129.4 (C-1, C-6 'd), 127.6 (C-5, s), 122.7 (C-6, d), 119.7 (C-α, d), 116.1 (C-5', d), 115.6 (C-3 ', d) , 110.9 (C-3 ", t), 101.1 (C-3, d), 56.0 (OCH 3), 40.8 (C-1", s), 27.6 (C-4 ", C-5", q) .

Value or a tin (Echinatin, compound 2): Yellow needle; UV λ _max (MeOH): 236, 312, 369 nm; EI-MS m / z : 270 [M] ⁺ (21), 239 (100), 121 (20); ¹ H-NMR (CD ₃ COCD ₃ , 500 MHz) δ 8.03 (1H, d, J = 15.5 Hz, H-β), 8.02 (2H, d, J = 8.5 Hz, H-2 ', H-6' ), 7.69 (1H, d, J = 8.0 Hz, H-6), 7.68 (1H, d, J = 15.5 Hz, H-α), 6.94 (2H, d, J = 8.5 Hz, H-3 ', H-5 '), 6.55 ( 1H, s, H-3), 6.51 (1H, d, J = 8.0Hz, H-5), 3.95 (3H, s, OCH 3); ¹³ C-NMR (CD ₃ COCD ₃ , 125 MHz) δ 188.5 (C = O), 162.3 (C-4 ', s), 162.1 (C-4, s), 161.4 (C-2, s), 139.3 (C-β, d), 131.8 (C-1 ', s), 131.6 (C-2', C-6 ', d), 131.2 (C-6, d), 119.6 (C-α, d) , 116.8 (C-1, s), 116.7 (C-3 ', C-5', d), 108.9 (C-5, d), 99.9 (C-3, d), 56.0 (OCH ₃ ).

Rico chalcone D (Licochalcone D, compound 3): Yellow needle; UV λ _max (MeOH): 254, 359 nm; EI-MS m / z : 354 [M] ⁺ (9), 323 (100), 267 (12); ¹ H-NMR (CD ₃ COCD ₃ , 500 MHz) δ 7.95 (1H, d, J = 16.0 Hz, β), 7.93 (1H, brs, H-2 ′), 7.88 (1H, d, J = 8.5 Hz , H-6 '), 7.70 (1H, d, J = 16.0 Hz, H-α), 7.28 (1H, d, J = 8.0 Hz, H-6), 6.96 (1H, d, J = 8.5 Hz, H-5 '), 6.71 (1H, d, J = 8.0 Hz, H-5), 5.38 (1H, m, H-2 "), 3.86 (3H, s, OCH ₃ ), 3.39 (2H, d, J = 7.5 Hz, H-1 "), 1.74 (3H, s, H-4"), 1.73 (3H, s, H-5 "); ¹³ C-NMR (CD ₃ COCD ₃ , 125 MHz) δ 188.6 (C = O), 160.2 (C-4 ', s), 149.6 (C-4, s), 149.2 (C-2, s), 139.2 (C-3, s), 139.1 (C-β, d), 133.1 (C-1 ', s), 131.7 (C-6', d), 131.4 (C-3 ", s), 129.2 (C -3 ', s), 129.1 (C-2', d), 123.2 (C-2 ", d), 121.5 (C-1, s), 120.9 (C-α, d), 120.1 (C-6 , d), 115.6 (C-5 ', d), 112.5 (C-5, d), 61.7 (OCH ₃ ), 25.9 (C-1 ", t), 25.9 (C-5", q), 17.9 (C-4 ", q).

Rico chalcone A (Licochalcone A, Compound 4): Yellow powder; UV λ _max (MeOH): 378, 308 nm; EI-MS m / z : 339 [M + 1] ⁺ , 323, 307, 216, 192, 121; ¹ H-NMR (CD ₃ COCD ₃ , 500 MHz) δ 8.03 (1H, d, J = 15.5 Hz, H-β), 8.01 (2H, d, J = 8.5 Hz, H-2 ', H-6' ), 7.68 (1H, d, J = 15.5 Hz, H-α), 7.63 (1H, s, H-6), 6.95 (2H, d, J = 8.5 Hz, H-3 ', H-5') , 6.58 (1H, s, H-3), 6.30 (1H, dd, J = 17.5 and 11.0 Hz, H-2 "), 5.04 (1H, dd, J = 17.5 and 1.5 Hz, H-3"), 4.98 (1H, doublet of doublets, J = 11.0 and 1.5Hz, H-3 "), 3.86 (3H, s, OCH ₃ ), 1.51 (6H, s, H-4", H-5 "); ¹³ C-NMR (CD ₃ COCD ₃ , 125 MHz) δ 188.5 (C = O), 162.2 (C-4 ', s), 160.0 (C-4, s), 159.8 (C-2, s), 148.7 (C-2 ", d), 140.1 (C-β, d), 131.8 (C-1 ', s), 131.6 (C-2', C-6 ', d), 129.3 (C-6, d), 127.6 ( C-5 ', s), 119.6 (C-α, d), 116.4 (C-3', C-5 ', d), 115.9 (C-1, s) α, 110.1 (C-3 ", t ), 101.1 (C-3, d), 56.0 (OCH ₃ ), 40.7 (C-1 ", s), 27.5 (C-4", C-5 ", q).

Iso leakage utility angiogenin (Isoliquiritigenin, compound 5): Yellow needle; ESI-MS m / z : 257 [M + l] ⁺ , 255 [M 1] ⁻ ; ¹ H-NMR (CD ₃ COCD ₃ , 500 MHz) δ 13.60 (s, OH-2 '), 8.12 (1H, d, J = 8.5 Hz, H-6'), 7.84 (1H, d, J = 15.0 Hz , H-β), 7.76 (1H, d, J = 15.0 Hz, H-α), 7.73 (2H, d, J = 8.0 Hz, H-2, H-6), 6.93 (2H, d, J = 8.0 Hz, H-3, H-5), 6.47 (1H, d, J = 8.5 Hz, H-5 ′), 6.38 (1H, brs, H-3 ′); ¹³ C-NMR (CD ₃ COCD ₃ , 125 MHz) δ 192.9 (C = O), 167.6 (C-4 ', s), 165.6 (C-2', s), 161.0 (C-4, s), 145.2 (C-β, d), 133.4 (C-6 ', s), 131.8 (C-2, C-6, d), 127.7 (C-1, s), 118.4 (C-α, d), 116.8 (C-3, C-5, d), 114.6 (C-1 ', s), 108.7 (C-5', d), 103 8 (C-3 ', d).

Rico Agrobacterium chalcone A (Licoagrochalcone A, Compound 6): Yellow powder; UV λ _max (MeOH): 298, 382 nm; EI-MS m / z : 324 [M] ⁺ (73), 323 (31), 188 (32), 175 (100), 137 (87), 133 (51); ¹ H-NMR (CD ₃ COCD ₃ , 500 MHz) δ 8.10 (1H, d, J = 8.5 Hz, H-6 ′), 7.79 (1H, d, J = 15.5 Hz, H-β), 7.73 (1H , d, J = 15.5 Hz, H-α, 7.62 (1H, brs, H-2), 7.58 (1H, d, J = 8.5 Hz, H-6), 6.94 (1H, d, J = 8.5 Hz , H-5), 6.48 (1H, d, J = 8.5 Hz, H-5 '), 6.37 (1H, brs, H-3'), 5.37 (1H, m, H-2 "), 3.36 (2H , d, J = 7.2 Hz, H-1 "), 1.73 (6H, s, H-4", H-5 "); ¹³ C-NMR (CD ₃ COCD ₃ , 125 MHz) δ 192.8 (C = O), 167.3 (C-2 ', s), 165.6 (C-4', s), 158.7 (C-4, s), 145.6 (C-β, d), 133.3 (C-6 ', d), 132.9 (C-3 ", s), 131.9 (C-2, d), 129.7 (C-3, s), 129.3 (C -6, d), 127.7 (C-1, s), 123.4 (C-2 ", d), 118.1 (C-α, d), 116.3 (C-5, d), 114.5 (C-1 ', s), 108.7 (C-5 ', d), 103.8 (C-3', d), 29.1 (C-1 ", t), 25.9 (C-5", q), 17.9 (C-4 ", q).

5 frame nilbu heptane (5-Prenylbutein, Compound 7): Amorphous powder; UV λ _max (MeOH): 375 nm; HRMS m / z 341.1318 [M + 1] ⁺ (calcd for C ₂₀ H ₂₁ O ₅ 341.1389); ¹ H-NMR (CD ₃ COCD ₃ , 500 MHz) δ 13.99 (s, OH-2 ′), 7.98 (1H, d, J = 8.0 Hz, H-6 ′), 7.77 (1H, d, J = 15.0 Hz, H-β), 7.68 (1H, d, J = 15.0 Hz, H-α), 7.35 (1H, d, J = 2.1 Hz, H-2), 7.22 (1H, d, J = 2.1 Hz , H-6), 6.91 (1H, d, J = 8.5 Hz, H-5 '), 6.53 (1H, brs, H-3'), 5.27 (1H, m, H-2 "), 3.37 (2H , d, J = 7.0 Hz, H-1 "), 1.78 (3H, s, H-4"), 1.64 (3H, s, H-5 "); ¹³ C-NMR (CD ₃ COCD ₃ , 125 MHz) δ 193.1 (C = O), 164.8 (C-2 ', s), 162.6 (C-4', s), 149.1 (C-5, s), 146.4 (C-4, s), 145.4 (C-β, d), 131.6 (C-6 ', d), 130.4 (C-3 ", s), 128.4 (C-3, d), 123.5 (C -2, d), 123.4 (C-2 ", d), 121.0 (C-1, s), 118.7 (C-α, d), 116.4 (C-1 ', s), 116.1 (C-5' , d), 116.0 (C-3 ', d), 114.5 (C-6, d), 25.9 (C-1 ", t), 22.3 (C-5", q), 18.6 (C-4 ", q).

Canzonol C (Compound 8 ): Yellow oil; UV λ _max (MeOH): 203, 340, 371, 375 nm; EI-MS m / z: 393 [M + 1] ⁺ (15), 392 [M] ⁺ (49), 349 (50), 337 (16), 307 (20), 293 (12), 281 (6 ), 205 (25), 187 (25); ¹ H-NMR (CD ₃ COCD ₃ , 500 MHz) δ 14.02 (s, OH-2 ′), 7.93 (1H, d, J = 8.5 Hz, H-6 ′), 7.82 (1H, d, J = 15.5 Hz, H-β), 7.72 (1H, d, J = 15.5 Hz, H-α), 7.61 (1H, brs, H-2), 7.56 (1H, d, J = 8.0 Hz, H-6) , 6.94 (1H, d, J = 8.5 Hz, H-5 '), 6.53 (1H, d, J = 8.0 Hz, H-5), 5.36 (1H, m, H-2 "), 5.28 (1H, m, H-2 '''), 3.36 (4H, d, J = 7.5 Hz, H-1 ", H-1'''), 1.78 (3H, s, H-4"), 1.74 (3H, s, H-4 '''), 1.72 (3H, s, H-5'''), 1.64 (3H, s, H-5 "); ¹³ C-NMR (CD ₃ COCD ₃ , 125 MHz) δ 193.1 (C = O), 164.8 (C-2 ', s), 162.6 (C-4', s), 158.6 (C-4, s), 145.4 (C-β, d), 132.9 (C-6 ', d), 131.8 (C-3 ", C-3''' s), 130.3 (C-2, d), 129.7 (C-3, s), 129.5 (C-3 ', s), 129.2 (C-6, d), 127.8 (C-1, s), 123.4 (C-2 ", d), 123.3 (C-2''', d), 118.3 (C-α d), 116.3 (C-5, d), 114.5 (C-1 ', s), 108.0 (C-5', d), 25.9 (C-1 ", C-1 '''t), 22.4 (C-5 ", C-5''' q), 17.9 (C-4 '''q), 17.8 (C-4", q).

< Example  4: Preparation of influenza virus

4-1. Avian Influenza (H9N2) Virus

The avian influenza virus used in the experiment was a low-pathogenic avian influenza virus A / chicken / Korea / 01310/2001 (H9N2). This was inoculated into the 10-day-old SPF (Specific-Pathogen Free) fertilized egg Allantoic Cavity, and 2 days later, the virus was inoculated into MDCK (Madin-Darby canine kidney) belonging to kidney cells and incubated for 5 days, followed by centrifugation. The culture of the supernatant layer was then used to measure the activity of viral neuraminidase of H9N2 origin.

4-2. Swine Flu Virus (H1N1) Virus

The swine influenza virus used in the experiment was A / Sw / Kor / CAN1 / 04 (H1N1, KCTC 11165BP), a swine influenza virus from the Central Vaccine Institute. The virus was inoculated into 10-day-old Specific-Pathogen Free (SPF) Egg Allantoic Cavity and harvested 2 days later to propagate the seed virus. The cultured seed virus was then reinoculated into the lumen cavity of SPF eggs to obtain a virus culture, and then used to measure the activity of viral neuraminidase of H1N1 origin.

< Example  5: Neuraminidays Cloning >

The H1N1 virus is highly contagious and is highly likely to die of disease if infected. Therefore, the H1N1 virus is primarily used as a target for new drugs rather than identifying and identifying active substances that are effective. Only Days was expressed in animal cell lines to validate the activity of drug candidates. The neuraminidase gene sequence of the H1N1 virus was confirmed from the NCBI gene bank, and cloned the neuraminidity of the H1N1 virus, and artificially produced a neuraminidism point mutant of the H1N1 virus was resistant to Tamiflu. Was used to explore the activity of natural products. Hereinafter, each base sequence used for cloning is as follows.

Swine Flu Neuraminidase Peptide Sequences Used

5-1. Neuraminidase Peptide Sequences of Swine Flu (H1N1) Virus

MNPNQKIITIGSVCMTIGMANLILQIGNIISIWISHSIQLGNQNQIETCNQSVITYENNTWVNQTYVNISNTNFAAGQSVVSVKLAGNSSLCPVSGWAIYSKDNSVRIGSKGDVFVIREPFISCSPLECRTFFLTQGALLNDKHSNGTIKDRSPYRTLMSCPIGEVPSPYNSRFESVAWSASACHDGINWLTIGISGPDNGAVAVLKYNGIITDTIKSWRNNILRTQESECACVNGSCFTVMTDGPSNGQASYKIFRIEKGKIVKSVEMNAPNYHYEECSCYPDSSEITCVCRDNWHGSNRPWVSFNQNLEYQIGYICSGIFGDNPRPNDKTGSCGPVSSNGANGVKGFSFKYGNGVWIGRTKSISSRNGFEMIWDPNGWTGTDNNFSIKQDIVGINEWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPKENTIWTSGSSISFCGVNSDTVGWSWPDGAELPFTIDK

5-2. Neuraminidase Peptide of Swine Flu Virus Resistant to Tamiflu

Underlined part is changed by point mutation (H → Y)

MNPNQKIITIGSVCMTIGMANLILQIGNIISIWISHSIQLGNQNQIETCNQSVITYENNTWVNQTYVNISNTNFAAGQSVVSVKLAGNSSLCPVSGWAIYSKDNSVRIGSKGDVFVIREPFISCSPLECRTFFLTQGALLNDKHSNGTIKDRSPYRTLMSCPIGEVPSPYNSRFESVAWSASACHDGINWLTIGISGPDNGAVAVLKYNGIITDTIKSWRNNILRTQESECACVNGSCFTVMTDGPSNGQASYKIFRIEKGKIVKSVEMNAPNY Y YEECSCYPDSSEITCVCRDNWHGSNRPWVSFNQNLEYQIGYICSGIFGDNPRPNDKTGSCGPVSSNGANGVKGFSFKYGNGVWIGRTKSISSRNGFEMIWDPNGWTGTDNNFSIKQDIVGINEWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPKENTIWTSGSSISFCGVNSDTVGWSWPDGAELPFTIDK

After cloning each gene with the above sequence, a PCR amplification method was performed on the gene corresponding to the neuraminidase of the H1N1 virus, and the amplified product was pcDNA3.1 / V5-His Topo vector of Invitrogen. Cloned into. Cloning was confirmed primarily through restriction enzyme cutting and finally confirmed by DNA sequencing. In addition, Neuraminidase resistant to Tamiflu was obtained by substituting the histidine amino acid with Tyrosine in the literature. The method used was a quick change method using PCR. This method produced an oligonucleotide corresponding to the portion to be replaced, and then amplified the plasmid in vitro using a PCR enzyme. DpnI enzyme that cleaves only the plasmids was treated. The mutated plasmid was amplified in bacteria and used for the experiment by finally confirming amino acid substitution through DNA sequencing.

< Example  6: Of neuraminidays  Active measurement>

The neuraminidase activity was used by modifying the method devised by Myers et al. In order to measure the exact inhibition rate. Each 20 μl of 0.04 M sodium acetate buffer (pH 5.0) and 80 μl of 0.04 mM 4-methylumbelliferyl-α-DN-acetylneuraminic acid (Sigma M8639) were mixed with each neuraminidase sample for 10 minutes, followed by 0.1 M After the reaction was stopped by adding 100 μl of glycine-NaOH buffer, activity was measured using a fluorescence difference at 360 nm / 440 nm using a fluorescence spectrometer. At this time, Tamiflu, the compound, or each solvent and fraction extract were pretreated in the cell or administered together when culturing the virus to be activated (in order to process the compound directly on the sample, 1 μl of the compound was added to the fluorescence reaction). ). In addition, in order to correct the luminescence of the sample, the inhibition rate was calculated using A as the measured luminescence, C as the luminescence of the solvent in which the sample was dissolved, and B as the luminescence of the sample mixture.

Inhibition Rate: {C- (A-B) / C} × 100

For kinetic studies of the compounds, the concentration of 4-methoxyumbelliferone was measured before adding 0.1M glycine-NaOH buffer to stop the reaction and calculated in Sigmaplot 11.0 (SPCC Inc., Chicago, IL). In addition, in order to measure the reversible reaction of the enzyme, it was measured using a method of diluting the reaction solution containing the enzyme and the inhibitor.

< Example  7: virus culture Of neuraminidays  Active measurement>

To measure neuraminidase activity of viral origin, a method of measuring neuraminidase activity from virus cultures derived from avian influenza virus (H9N2) and swine influenza virus (H1N1) was used.

The activity of neuraminidase using a virus culture was measured by the method of Example 6 using the virus cultures of Examples 4-1 and 4-2, and each fraction of the ethanol extract and neurai using Compound 1 to Compound 8 were used. As a result of measuring the activity of mini-day is shown in Table 2 and Table 3. As shown in Table 2, in order to compare the activity of ethanol extracts and solvent fractions against neuraminidase, the extract activity of each extract against neuraminidase obtained from virus culture at the final concentration of 20 µg / ml. Was measured. As can be seen from the results shown in Table 2, the extract was confirmed to have excellent activity in the 70% ethanol extract and ethyl acetate fraction.

 Condition Inhibitory Activity of Avian Influenza Virus (H9N2) on Neuraminidase (20 μg / mL) Inhibitory Activity of Porcine Influenza Virus (H1N1) on Neuraminidase (20 μg / mL)  A fraction (70% ethanol extract)  36.2%  37.6%  B fraction (hexane extract)  -  -  C fraction (ethyl acetate extract)  28.5%  32.7%  D fraction (butanol extract)  -  -

As shown in Table 3, Tamiflu showed that the inhibitory activity (12.05 ± 1.00 nM) against neuraminidase of avian influenza virus origin was better than the inhibitory activity against neuraminidase of swine influenza virus origin (96.89 ± 7.41 nM). I could confirm it. These results illustrate that Tamiflu acts selectively on avian influenza and may be less active against swine flu or swine flu. However, compounds 1 to 8 of the present patents were found to inhibit both neuraminidase of avian influenza and swine influenza virus origin at similar concentrations as shown in Table 3.

Tamiflu is made by synthesizing compounds that selectively bind to active residues after confirming the neuraminidase protein structure of avian influenza. Tamiflu acts as an active residue as a competitive inhibitor of neuraminidase. However, recently, viruses resistant to Tamiflu have obtained resistance by using a method of modifying the active residue on which Tamiflu acts. The inventors used one of the active compounds to determine the inhibition pattern of the chalcone family of compounds. Inhibitory form was examined using a method of varying the concentration of Lycochalcone A (Compound 4 ). Lycokalkin A (Compound 4), one of the main compounds of licorice, reversibly inhibits neuraminidase of swine influenza virus origin as a noncompetitive inhibitor. These results confirm that the chalcone-based compound acts non-competitively on neuraminidase and thus can be widely used alone or in combination with Tamiflu despite the emergence of resistant viruses.

 Condition IC ₅₀ Measurements for Neuraminidase of Avian Influenza Virus (H9N2) IC ₅₀ Measurements for Neuraminidase of Swine Influenza Virus (H1N1) Compound 1 (μg / ml)  52.16 ± 11.12  37.68 ± 3.18 Compound 2 (μg / ml)  5.70 ± 0.55  5.81 ± 0.41 Compound 3 (μg / ml)  35.22 ± 8.21  28.62 ± 5.68 Compound 4 (μg / ml)  17.99 ± 0.98  19.10 ± 2.10 Compound 5 (μg / ml)  9.70 ± 0.38  8.41 ± 0.39 Compound 6 (μg / ml)  56.92 ± 3.12  51.59 ± 2.77 Compound 7 (μg / ml)  35.50 ± 1.43  25.87 ± 2.04 Compound 8 (μg / ml)  39.96 ± 0.59  75.38 ± 14.47  Tamiflu (nM)  12.05 ± 1.00  96.89 ± 7.41

< Example  8 : Neuraminidays  In the lysate of cells that express directly Of neuraminidays  Active measurement>

In order to measure the activity of neuraminidase in cell disruption cells expressing the neuraminidase of the H1N1 virus and the Tamiflu-resistant H1N1 virus, human kidney kidney HEK293T cells were treated with 0.25% trypsin and collected. The supernatant was removed and then suspended in DMEM medium free of bovine serum. 3 μl of lipofectamine (Lipofectamin, Invitrogen, Inc) was added to 100 μl of DMEM without bovine serum, and the mixture was slowly mixed and allowed to stand at room temperature for 15 minutes. Subsequently, transfection of the plasmids expressing the neuraminidase of the swine flu virus (Example 5-1) and the neuraminidase of the Tamiflu-resistant swine flu virus (Example 5-2) was performed by transfection (DNA) into animal cells. Method), each plasmid of 1 µg was slowly added dropwise to the microneedle tube over 30 seconds, and left for 15 minutes at room temperature. The mixture of lipofectamine and DNA-plasmid and HEK293T cell line were slowly mixed and incubated at room temperature in suspension for 20 minutes, then aliquoted in a 35 mm culture vessel and incubated in a CO ₂ incubator for 24 hours. HEK293T cells were washed twice with PBS, 500 μl of extract solution was added to each culture vessel, and the cells were disrupted and centrifuged at 14,000 rpm for 5 minutes. The obtained supernatant was purified by using the method of Example 6 Used for activity experiment.

Table 4 shows compounds 2 (Echinatin), which are active and active against neuraminidase of viral origin, among the chalcone compounds 1 to 8 isolated from licorice extract, It is the result of having measured the activity with respect to the neuraminidase of the swine flu virus and the neuraminidase of the Tamiflu-resistant swine flu virus about the compound 4 (Ricochalcon A) and the compound 5 (Isocristygenin). Measurement result chalcone-based compound 2 (Echinatin), Compound 4 (Ricocalcon A) and Compound 5 (Isoricrigeninine) were similar concentrations of 2.49 ± 0.14 to 5.42 ± 0.28 μg / ml for the neuraminidase of the H1N1 virus and neuraminidase of the Tamiflu-resistant H1N1 virus. Activity was inhibited by 50%. However, Tamiflu showed an IC ₅₀ value at about 75 nM against neuraminidase of swine flu virus, but Tamiflu was less active to show IC ₅₀ value at 20 μM against artificially generated neuraminidase of Tamiflu resistant flu. . This shows the risk in the development of neuraminidase of the Tamiflu-resistant H1N1 virus, including chalcone compound 2 (echinatin) from licorice, Compound 4 (Ricochalcon A) and Compound 5 (Isocristygenin) show the potential for use against neuraminidase of the H1N1 virus and neuraminidase of the Tamiflu-resistant H1N1 virus.

 Condition IC ₅₀ for neuraminidase of novel influenza virus IC ₅₀ for swine flu neuraminidase of Tamiflu resistant virus  Compound 2 (μg / ml)  3.48 ± 0.05  3.42 ± 0.56  Compound 4 (ug / ml)  2.49 ± 0.14  2.19 ± 0.06  Compound 5 (ug / ml)  5.42 ± 0.28  4.20 ± 0.12  Tamiflu  75nM  > 20 μM

< Example  9: virus culture Tamifluwa Echinanthin  In combination use Of neuraminidays  Active measurement>

The 50% inhibition rate of Tamiflu against neuraminidase of the swine influenza virus identified in Example 7 was 96.89 ± 7.41nM, and the 50% inhibition rate of Tamiflu against neuraminidase of the avian influenza virus was 12.05 ± 1.00nM and Tamiflu was a competitive inhibitor It was confirmed. As a result confirmed in Example 7 Echinatin (Compound 2 ) is one of the compounds of the present invention is a non-competitive inhibitor (noncompetitive inhibitor, see Figure 2) shows a concentration of 50% inhibition against swine influenza 19.09 ± 2.10㎛ / Ml. Competitive inhibitors act at the active pocket where the neuraminidase enzyme binds to the compound, so adding non-competitive inhibitors of echinatin (compound 2 ) acting at other sites of the neuraminidase enzyme increases Tamiflu activity. The inhibition rate of Tamiflu was calculated using echinatin (Compound 2 ) under the assumption that it could be.

To this end, the concentration of echinatin (compound 2 ) from licorice was fixed at 5 μM, and then the virus cultures of Examples 4-1 and 4-2 were used for the neuraminidase of swine influenza virus and avian influenza virus origin. As a result of measuring the 50% inhibition rate of Tamiflu by the method of Example 6, the 50% inhibition rate of Tamiflu against swine influenza virus was measured to be 4.91 ± 1.81nM, and the activity of Tamiflu when Tamiflu and Echinatin (Compound 2 ) were used in combination was weak. It could be increased 20-fold, and about 10-fold increased activity for avian influenza virus. Currently recommended treatment for the pandemic of swine flu or avian influenza, Tamiflu alone, Tamiflu and amantadine (primarily rimantidine, rimantidine), Jannavir, etc., and the Amantadine series are highly toxic. Therefore, the combination therapy of chalcone-based compounds and Tamiflu is an effective strategy for the treatment of resistant virus. Table 5 shows the inhibition rate of neuraminidase activity of avian influenza and swine influenza viruses according to Tamiflu concentration (nM) in parallel with 5 μM of echinatin (Compound 2 ).

 Neuraminidays Types  Tamiflu concentration in parallel with 5 μM echinatin  0.12 nM  0.48 nM  1.9 nM  7.8 nM  31.3 nM  125 nM  Inhibition of Neuraminidase Activity of Avian Influenza Virus (H9N2) (%)  30.1  46.1%  54.5%  65.7%  78.7%  96.9%  Inhibition of Neuraminidase Activity of Porcine Influenza Virus (H1N1) (%)  23.6%  30.5%  45.8%  58.7%  65.7%  79.5%

< Example  10: hemagglutination test for avian and swine influenza viruses>

In order to measure the antiviral activity of the isolated compounds was measured using avian influenza virus (H9N2) and porcine influenza virus (H1N1). Subdivided MDCK (Madin-Darby canine kidney) cells belonging to renal cells, suspended at 300,000 cells / ml, and 100 μl / well per well were dispensed into 96-well plates, followed by incubation for 1 day in a 37 ° C. CO ₂ incubator. It is then infected with avian and swine influenza virus diluted to 10 ⁴ TCID ₅₀ / ml and reacted for 1 hour in a 37 ℃ CO ₂ incubator. Thereafter, the infected cells were washed three times with PBS, and then the compounds were diluted to a final concentration of 10 µg / ml, respectively, divided into three wells, and incubated in a 37 ° C. CO ₂ incubator for 5 days. Tamiflu was added at the same concentration as a positive control, and the negative control was only evaluated by infecting avian and swine influenza viruses. When the virus adheres to the host cell, specific erythrocytes are aggregated, and thus, the activity is evaluated by performing a hemagglutination assay (HA), which is one of the methods of indirectly quantifying the virus present in the cell culture. .

Table 6 shows the results of the erythrocyte agglutination test, chalcone-based compound 2 (Echinatin), which inhibits neuraminidase, It was confirmed that Compound 4 (Ricochalcon A) and Compound 5 (Isoricristinein) inhibited hemagglutination, and Tamiflu also inhibited hemagglutination.

 Condition  Antiviral Activity of Compounds by Hemagglutination  Avian Influenza Virus (H9N2)  Swine Influenza Virus (H1N1)  A fraction (ethanol extract)  None (+)  None (+)  B fraction (hexane extract)  none(-)  none(-)  C fraction (ethyl acetate extract)  Yes (+)  Yes (+)  D fraction (butanol extract)  none(-)  none(-)  Compound 2  Yes (+)  Yes (+)  Compound 4  Yes (+)  Yes (+)  Compound 5  Yes (+)  Yes (+)  Tamiflu  Yes (+)  Yes (+)

< Example  11: Toxicity test

11-1. Acute Toxicity

This study was conducted to investigate the toxicity of acutely (within 24 hours) to the animal body and to determine the mortality rate when excessive intake of 70% ethanol extract and ethyl acetate activity fraction extracted from licorice. Twenty ICR mouse strains, which are common mice, were assigned to 10 control groups and 10 experimental groups. Only 30% PEG-400 was administered to the control group, and the experimental group was orally administered with an ethyl acetate activity fraction extracted from licorice at a concentration of 2.0 g / kg (about 50 times the amount used in general animal experiments). As a result of investigating each mortality after 24 hours, all of the control group and the experimental group administered the ethyl acetate activity fraction containing the licorice 70% ethanol extract and the compounds 1 to 8 at 2.0 g / kg concentration survived.

11-2. Long-term and tissue toxicity experiments in experimental and control groups

In the long-term toxicity experiment, C57BL / 6J mice were treated with 70% ethanol extract containing Compound 1 to Compound 8 and the control group only solvent to examine the effect on each organ (tissue) of the animal. Blood was collected after 8 weeks from the blood concentrations of glutamate-pyruvate transferase (GPT) and blood urea nitrogen (BUN) were measured using a Select E (Vital Scientific NV, Netherland) instrument. As a result, in the case of GPT, which is known to be related to hepatotoxicity, and BUN, which is known to be related to nephrotoxicity, there was no difference in the experimental group compared to the control group. In addition, liver and kidneys were removed from each animal, and histological observations were performed by optical microscopy through a conventional tissue fabrication process. No abnormalities were observed.

< Example  1: Pharmaceutical Formulation example >

1-1. Manufacture of tablets

Example 1 according to the present invention 200 g of 70% ethanol extract of licorice or a compound isolated therefrom was mixed with 175.9 g lactose, 180 g potato starch and 32 g colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.

1-2. Preparation of Injection

Example 1 according to the present invention 1 g, 70 g ethanol extract of licorice or a compound isolated therefrom, 0.6 g sodium chloride and 0.1 g ascorbic acid were dissolved in distilled water to make 100 ml. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

< Example  2: food Production Example >

2-1. Preparation of Cooking Seasonings

Licorice 70% ethanol extract of Example 1 according to the present invention 0.2 to 10 parts by weight of each compound isolated from it was prepared for health promotion cooking seasoning.

11-2. Manufacture of Flour Food

Licorice 70% ethanol extract of Example 1 according to the present invention or 0.1 to 5.0 parts by weight of each compound isolated therefrom is added to the flour, using the mixture to prepare bread, cake, cookies, crackers and noodles for health promotion Food was prepared.

2-3. soup  And juicy ( gravies Manufacturing

The licorice 70% ethanol extract of Example 1 according to the present invention, or 0.1 to 1.0 parts by weight of each of the compounds separated therefrom were added to soups and broth to prepare meat products for health promotion, soups and broths of noodles.

2-4. dairy product( dairy products Manufacturing

Licorice 70% ethanol extract of Example 1 according to the present invention, or 0.1 to 1.0 parts by weight of each compound isolated therefrom was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

< Example  3: drink Production Example >

3-1. Vegetable Juice  Produce

0.5 g of each of the licorice 70% ethanol extract of Example 1 or the compound isolated therefrom was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice for health promotion.

3-2. Fruit juice  Produce

Health promotion fruit juice was prepared by adding 0.1 g of the licorice 70% ethanol extract of Example 1 or the compound isolated therefrom to 1,000 ml of apple or grape juice according to the present invention.

< Example  4: feed additive Production Example >

10.0 g of each of the licorice 70% ethanol extract of Example 1 or a compound isolated therefrom according to the present invention was prepared by mixing an excipient with 50.0 g of a feed additive. However, the compounding ratio may be arbitrarily modified, and the above components may be mixed and used in production according to a conventional feed composition production method.

< Example  5: Preparation of Disinfectants>

Licorice 70% ethanol extract of Example 1 according to the present invention or 3.0 g of each compound isolated therefrom was added, and purified water was added to 1,000 ml to prepare a natural disinfectant for preservatives.

Figure 1 shows the HPLC analysis spectrum showing the peak and the compound appearing on the HPLC after confirming the structure by separating the active chalcone-based compounds isolated from licorice.

FIG. 2 shows that Echinatin (Compound 2 , compound concentration in μM) inhibits neuraminidase of swine influenza virus origin as a noncompetitive inhibitor.

Claims (12)

Licorice ( Glycyrrhiza uralensis ) is extracted with at least one extraction solvent selected from ethanol or methanol or an aqueous solution of these extraction solvents, and is a chalcone compound, such as ricochacone F, echinnatin, and ricochalcon D. , At least one of the compounds of Licochalcone A, Isoliquiritigenin, Licoagrochalcone A, 5-Prenylbutein, Kanzonol C Licorice extract for the prevention or treatment of avian and swine influenza and swine flu-related diseases, characterized in that it contains more than a branch. The method of claim 1, Extraction solvent is licorice extract for the prevention or treatment of avian and swine influenza and swine flu-related diseases, characterized in that 50 to 100% ethanol aqueous solution. A dietary supplement for the prevention or improvement of avian and swine influenza and swine flu-related diseases, comprising the licorice extract of claim 1.        The method of claim 3, wherein The health food from the group consisting of nutritional products, including drinks, meat, sausages, bread, candy, snacks, noodles, dairy products including ice cream, beverages, soups, ionic beverages, alcoholic beverages and vitamin complexes Health functional food, characterized in that selected. A pharmaceutical composition for preventing or treating avian and swine influenza and swine flu-related diseases, comprising the licorice extract of claim 1 as an active ingredient and containing a pharmaceutically acceptable carrier or excipient. delete A pharmaceutical composition for preventing or treating avian and swine influenza and swine flu-related diseases, comprising the licorice extract of claim 1 and Tamiflu as an active ingredient and a pharmaceutically acceptable carrier or excipient. Algae characterized in that it comprises at least one or more of the compounds of ricokalcon F, echinatin, ricochalcon D, ricokalcon A, isosricrigenin, ricoagrocalcon A, 5-prenylbutane, canzonol C and A pharmaceutical composition for preventing or treating swine influenza and swine flu-related diseases. delete A pharmaceutical composition for preventing or treating avian and swine influenza and swine flu-related diseases, comprising the compound of claim 8 and Tamiflu as an active ingredient and a pharmaceutically acceptable carrier or excipient. Animal drug and feed additive for the prevention or treatment of avian and swine influenza and swine flu disease comprising the licorice extract of claim 1 as an active ingredient and a pharmaceutically acceptable carrier or excipient. A natural antiseptic agent for preventing or treating avian and swine influenza and swine flu-related diseases, comprising the licorice extract of claim 1 as an active ingredient and a pharmaceutically acceptable carrier or excipient.

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