KR102456749B1 - Composition for preventing or treating swine influenza virus disease comprising Curcuma longa extract or sesquiterpenoids isolated therefrom as active ingredients - Google Patents

Abstract

The present invention is turmeric ( Curcuma longa ) extract or sesquiterpenoid compounds isolated therefrom, isoprocurcumenol (Compound 1), intermedin B (Compound 2), curcumadione (Compound 3), (-)-curcumenone (Compound 4) and (+ )- It relates to a composition for preventing and treating swine influenza comprising turmeronol A (Compound 5).

Description

A composition for preventing or treating swine influenza virus infection comprising an extract of turmeric (Curcuma longa) or a sesquiterpenoid-based compound isolated therefrom as an active ingredient TECHNICAL FIELD [0002] isolated therefrom as active ingredients}

The present invention relates to a composition for preventing, improving or treating swine influenza containing an extract and fractions or constituent compounds of Curcuma longa , which exhibits cell lesion recovery ability against swine influenza (H1N1) virus, and the same as an active ingredient, more In detail, the prevention of swine influenza, including any one of sesquiterpenoid-based compounds (isoprocurcumenol, intermedin B, curcumadione, (-)-curcumenone, (+)-turmeronol A) obtained from turmeric extract, and It relates to a therapeutic composition.

In the current animal breeding, a corporate-type livestock system that breeds a large number of breeding individuals in a dense environment is introduced, and genetically excellent individuals suitable for mass production are selected, produced through inbreeding, and then clustered and raised. However, mass breeding of genetically non-diverse individuals is a situation that not only intensively exposes to specific diseases due to low genetic diversity, but also spreads infectious diseases due to the dense breeding environment, affecting even humans. In addition, frequent contact between humans and animals and the increase in social interaction due to the development of transportation systems enabled the rapid spread of zoonotic diseases. In other words, until the development of transportation systems, diseases that occurred in a specific area were prevented from spreading due to the restriction of movement, which allowed humans to prepare for a minimum amount of time. There is a need for an integrated preparation to block the possibility of developing common diseases.

Influenza virus is an RNA virus (Orthomyxovirus) belonging to the ‘Orthomyxovirus’ and is classified into A, B, and C types among five genera. Type A is a virus that mainly infects humans, and is confirmed to be infected in pigs, other mammals, and various wild birds compared to types B and C. Recently, avian influenza, swine influenza, and novel flu, which have become a worldwide problem, all belong to type A viruses.

There are two glycoproteins, hemagglutinin (HA) and neuraminidase (NA), on the surface of the influenza virus. There are 16 types of HA and 9 types of NA, so 16×9=144 types of influenza Viruses may occur. Only three types of hemagglutinin (H1, H2, H3) and two types of neuraminidase (N1 and N2) are known to cause influenza infection in humans. Cases of transmission to humans have also occurred.

Swine influenza virus was first reported in 1918, and usually pigs have human influenza receptors (N-acetylneuraminic acid α2,6 galactose) and birds have influenza receptors (N-acetylneuraminic acid α2,3). galactose), so that the avian influenza virus can infect humans through recombination or adaptation in the pig's body is known to act as virus mixing vessels.

H1N1, a swine influenza virus, was confirmed to have mutated into a disease that was contagious to humans by infecting pigs with the novel flu, which first broke out in Mexico in 2009 and spread to the world, causing numerous deaths. As of early October 2009, the number of infected people of the H1N1 flu, as compiled by the World Health Organization (WHO), reached 343,200 and the death toll exceeded 4,000. It responded by upgrading it to the 6th stage, the pandemic state. H1N1 flu has symptoms of fever, sore throat, cough, runny nose, or stuffy nose similar to the general flu, but furthermore, it causes more fatal lung damage than the general flu. there was. Therefore, it is essential to secure a vaccine to prevent H1N1 influenza, and it is necessary to produce and inoculate the vaccine nationally before H1N1 flu spreads rapidly. However, as a preventative national vaccine measure, it is almost impossible to make a vaccine that prevents all RNA viruses that cause antigenic mutations every time, and it takes several months to supply a mass vaccine. the reality is that it is not. In addition, in order to prepare for the occurrence of mutant pathogens such as avian and swine influenza, swine flu, or killer flu, which is a mixture of avian influenza and swine flu, which are the worst possible outbreaks, it is essential to develop a therapeutic agent that can prevent and treat them.

Until now, development of therapeutic agents for various target points based on influenza A virus proliferation and pathological infection mechanisms have been attempted. In Korea, there is an inhaled form of zanamivir that was developed and marketed in 2001 in the same way as the oral treatment Tamiflu (oseltamivir phosphate) marketed in 2001 in Korea. Others include Xofluza (baloxabir), an oral treatment that was approved for domestic sales in 2019 and is scheduled to be released soon. As side effects of Tamiflu, which has monopolized the flu treatment market for the past 20 years, nausea, vomiting, and nervous or mental disorders have been reported. . In addition, the appearance of a virus resistant to Tamiflu has been reported, and even human-to-human transmission of the Tamiflu-resistant virus has been found. Therefore, the development of a virus treatment for avian and swine influenza and swine flu is essential for human health and safety. need more

Looking at the process of infection and proliferation of viruses, viruses produce a new virus within the cell after infection, and in order to spread to other cells, sialic acid existing outside the cell is cut using neuraminidase made by the virus. something is needed Tamiflu-resistant avian influenza virus is known to occur through three genetic mutations (Arg292Lys, Asn274Ser, His295Tyr) that change the amino acid of the Tamiflu receptor protein to which Tamiflu binds. Among the three genetic mutations, in the case of the Tamiflu-resistant avian influenza virus, in which His at 295 is changed to Tyr, which is expressed the most, the hydrophobic residue of Tamiflu is mutated to prevent binding to the Tamiflu receptor. . This virus was discovered with a 10-25% probability in the 2007-2008 season in the United States and Europe, increasing the risk of mutagenicity to the H1N1 influenza in the future. Therefore, the emergence of these Tamiflu-resistant viruses provides justification for humans to continuously develop new viral therapeutics.

In the present invention, in consideration of the urgency of the protective measures against swine influenza and swine flu, substances that recover cellular lesions caused by swine influenza were searched for food and medicinal plants that can be adapted immediately when the effect is confirmed. In general, there are many advantages to finding new active ingredients from natural drugs used in traditional medicine rather than experimentally modifying existing drugs to develop new drugs. In particular, since these active ingredients have been used for a long time, there is little concern about toxicity caused by drugs. This fact can be confirmed from the fact that the starting material for Tamiflu, which is used as a treatment for the H1N1 influenza, was developed from the fruit of 'Star Anis', a native plant in China, as the main raw material.

The subject of this patent, turmeric ( Curcuma longa ) is a perennial herb native to India and widely cultivated in tropical and subtropical regions from India to Southeast Asia. Turmeric is a plant that is recently cultivated in the southern regions of Korea and is used as a raw material for curry and the like. Turmeric contains 1 to 5% of aromatic essential oil and 0.3% of curcumin, a yellow pigment, so it has been used as a flavoring agent for hepatitis, cholangitis, cholelithiasis, and catarrhal jaundice.

The present inventors have completed the present invention by confirming that the sesquiterpenoid-type compound isolated from turmeric has the effect of restoring cell lesions caused by swine influenza virus while researching the compound having the pharmacological activity of turmeric.

In Korean Patent No. 1373219, it was confirmed that the fraction had antiviral activity against influenza virus H1N1 after methanol extraction of kandonghwa flower, passerine seed, astragalus root, and sage plant. Although the anti-influenza virus agent containing polis, xylitol, frankincense, myrrh, passerine, doin, samreung, bongchul and vitamin C as active ingredients is described, pig containing a sesquiterpenoid-type compound isolated from turmeric of the present invention It is different from the composition for preventing or treating influenza virus infection.

Korean Patent Application Laid-Open No. 20030014556 discloses Curcuma xanthorrhiza ) isolated from xanthorrhizol and disclosed an antiviral agent having antiviral activity against HIV virus, herpes II virus, and influenza A virus, but a sesquiterpenoid family isolated from turmeric of the present invention It is different from the composition for preventing or treating swine influenza virus infection comprising a compound of

Korean Patent No. 1077920 is Curcuma Longa longa ) discloses activity against H1N1 influenza virus of three types of curcuminoids (curcumin, demethoxycurcumin, bisdemethoxycurcumin) isolated from ), but this is also a sesquiterpe isolated from turmeric of the present invention It is different from the composition for preventing or treating swine influenza virus infection including a compound of the noid series. In addition, Korean Patent Application Laid-Open No. 20070102487 discloses a first ingredient obtainable from ginger; a second component obtainable from green tea; any third ingredient obtainable from turmeric; And a composition for reducing the transmission rate of a disease comprising administering to a mammal or a bird a certain amount of the composition having an acceptable carrier is a herpes virus, HIV virus, AIDS virus, influenza virus, H5N1 influenza virus, nasal cold virus, SARS Although it is described that it is effective against viruses selected from the group consisting of viruses and respiratory syncytial viruses, a composition for preventing or treating swine influenza virus infection comprising a sesquiterpenoid-type compound isolated from turmeric of the present invention is different from

Korean Patent No. 1373219, medicinal plant extract having antiviral activity against influenza virus H1N1, registered on 03.05.2015. Korean Patent No. 1317318, anti-influenza virus agent, 2016.05.27. registration. Korean Patent Application Laid-Open No. 20030014556, novel antiviral agent, published on February 19, 2003. Korean Patent No. 1077920, a composition for preventing and treating influenza virus infection containing turmeric extract and a composition for inhibiting neuraminidase activity, registered on October 24, 2011. Korean Patent Application Laid-Open No. 20070102487, Composition and method for reducing disease transmission rate, published on October 18, 2007.

Dong J.Y., et al., Sesquiterpenoids from Curcuma wenyujin with anti-influenza viral activities, Phytochemistry, 85, 122-128, 2013. Lee J.Y., The resistance situation of antiviral agent in Korea: 2008-2009 oseltamivir resistance of seasonal influenza A/H1N1, Infect. Chemother., 41 (Suppl 2), S20-S24, 2009. Schnitzler S.U., et al., An update on swine-origin influenza virus a/h1n1: A review, Virus Genes, 39(3), 279-292, 2009. Wright P.F., et al., Orthomyxoviruses, Fields' Virology, 5th ed. New York: Wolfers Kluwer Health, Lippincott Williams & Wilkins, 1691-1740, 2007. Zimmer S.M., et al., Historical perspective-emergence of influenza A (H1N1) viruses, N. Engl. J. Med., 361, 279-285, 2009. Dawood F.S., et al., Emergence of a novel swine-origin influenza A (H1N1) virus in humans, N. Engl. J. Med., 360, 2605-2615, 2009. Hauge S.H., et al., Oseltamivir-resistant influenza viruses A (H1N1), Norway, 2007-2008, Emerg. Infect. Dis., 15, 155-162, 2009

The object of the present invention is turmeric ( Curcuma longa ) to provide a composition for preventing or treating swine influenza virus infection, characterized in that it contains an extract or a sesquiterpenoid-based compound isolated therefrom as an active ingredient.

An object of the present invention is a pharmaceutical composition for the prevention or treatment of swine influenza virus infection, comprising, as an active ingredient, sesquiterpenoid-type compounds obtained by pure separation and purification from turmeric, which has the effect of recovering cell lesions caused by the swine influenza virus. is to provide

Another object of the present invention is turmeric ( Curcuma longa ) extract or a sesquiterpenoid-based compound isolated therefrom as an active ingredient to provide a health functional food for preventing or improving swine influenza virus infection.

Another object of the present invention is turmeric ( Curcuma longa ) extract or to provide an animal drug for the prevention or treatment of swine influenza virus infection comprising a sesquiterpenoid-based compound isolated therefrom as an active ingredient.

Another object of the present invention is turmeric ( Curcuma longa ) extract or to provide an animal feed additive for prevention or improvement of swine influenza virus infection comprising a sesquiterpenoid-based compound isolated therefrom as an active ingredient.

In addition, the object of the present invention is turmeric ( Curcuma longa ) extract or a sesquiterpenoid-based compound isolated therefrom to provide a natural disinfectant for preventing swine influenza virus infection as an active ingredient.

The present invention relates to a composition for preventing or treating swine influenza virus infection, comprising an extract of Curcuma longa or a sesquiterpenoid-based compound isolated therefrom as an active ingredient.

The turmeric extract is

(a) extracting turmeric with 40-90% (v/v) ethanol and concentrating it under reduced pressure;

(b) suspending the ethanol extract concentrated under reduced pressure of step (a) in distilled water; and

(c) solvent-extracting the suspension of step (b) with ethyl acetate; 40-90% (v/v) ethanol-ethyl acetate fraction of turmeric obtained through a turmeric extract separation method comprising a.

The 40-90% (v/v) ethanol-ethyl acetate fraction of turmeric may contain 50%-99% of sesquiterpenoid compounds.

In addition, the sesquiterpenoid-based compound is isoproculcumenol (Compound 1), intermedin B (Compound 2), curcumadione (Compound 3), (-)-curcumenone (Compound 4) of Formula 1 below. and (+)-talmeronol A (Compound 5). It may include at least one compound selected from the group consisting of.

[Formula 1]

In addition, the present invention provides isoproculcumenol (Compound 1), Intermedin B (Compound 2), Curcumadione (Compound 3), (-)-Curcumenone (Compound 4) and (+)-hair of Formula 1 above Curcuma containing at least one compound selected from the group consisting of Meronol A (Compound 5) longa ) relates to an animal drug for preventing or treating swine influenza containing an extract as an active ingredient.

The turmeric extract may be an extract obtained by extracting turmeric with one or more solvents selected from the group consisting of water, C1 to C4 lower alcohol, acetone, ethyl acetate and hexane, and the C1 to C4 lower alcohol is methanol, ethanol , propanol, isopranol, butanol, and the like. Preferred are ethanol, butanol and acetone. Most preferred are ethanol and butanol. The solvent may be added by 5 times to 500 times the weight of turmeric. In addition, the turmeric extract is a diluted solution obtained by concentrating the extract obtained by extracting turmeric with one or more solvents selected from the group consisting of water, C1 to C4 lower alcohol, acetone, ethyl acetate and hexane. It may be a fraction fractionated by being added to the charged column chromatography and eluting with an organic solvent. The dilution may be made by adding water to the concentrate of the turmeric extract, and 10 to 50 times the weight of the concentrate may be added. The ion exchange resin may be selected from Diaion HP-20 (Diaion HP-20), SP825, AXT204, XAD1600T, MN200, SP70, SP710, and the like. Preferred are Diaion HP-20, SP825, AXT204, XAD1600T and MN2000, and most preferably Diaion HP-20. The organic solvent may be a C1 to C4 lower alcohol and acetone. The C1 to C4 lower alcohol may be methanol, ethanol, propanol, isopranol, butanol, or the like. Preferred are ethanol and acetone, most preferably 100% ethanol.

Isoproculcumenol (Compound 1), Intermedin B (Compound 2), Curcumadione (Compound 3), (-)-Curcumenone (Compound 4) and (+)-Tulmero of Formula 1 isolated from the turmeric Nol A (Compound 5) compound can be obtained by fractionation of turmeric extract by chromatography, and the chromatography is Diaion HP-20 column chromatography, silica gel column chromatography ), RP-18 column chromatography, LH-20 column chromatography, preparative reversed-phase high performance chromatography, medium pressure liquid Chromatography (medium pressure liquid chromatography), high-performance liquid chromatography (high-performance liquid chromatography), etc. can be selected and used.

Meanwhile, the compound of the present invention may be synthesized according to a conventional method in the art, and may be prepared as a pharmaceutically acceptable salt.

The pharmaceutical composition comprising the turmeric extract or the sesquiterpenoid-based compound isolated therefrom, respectively, is a powder, granules, tablets, capsules, suspensions, emulsions, syrups, oral dosage forms, such as aerosols, according to a conventional method; It can be used in the form of external preparations, suppositories and sterile injection solutions. Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient, e.g. For example, it is prepared by mixing starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, etc. may be used.

The dosage of the pharmaceutical composition will vary depending on the age, sex, and weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration, and the judgment of the prescriber. Dosage determination based on these factors is within the level of one of ordinary skill in the art, and generally dosages range from 0.01 mg/kg/day to approximately 2000 mg/kg/day. A more preferred dosage is 0.1 mg/kg/day to 500 mg/kg/day. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way. The pharmaceutical composition may be administered to mammals such as mice, livestock, and humans by various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine mucosal or intracerebrovascular injection. The turmeric extract of the present invention or the sesquiterpenoid-based compound isolated therefrom has almost no toxicity and side effects, so it is a drug that can be safely used even when taken for a long period of time for prophylactic purposes.

In addition, the present invention provides isoproculcumenol (Compound 1), Intermedin B (Compound 2), Curcumadione (Compound 3), (-)-Curcumenone (Compound 4) and (+)-hair of Formula 1 above Curcuma containing at least one compound selected from the group consisting of Meronol A (Compound 5) longa ) relates to a health functional food for preventing or improving swine influenza infection containing the extract as an active ingredient.

In addition, the present invention is isoproculcumenol (Compound 1), intermedin B (Compound 2), curcumadione (Compound 3), (-)-curcumenone (Compound 4) and ( It relates to a health functional food for preventing or improving swine influenza virus infection containing at least one compound selected from the group consisting of +)-talmeronol A (compound 5) as an active ingredient.

The present invention provides a health functional food comprising a nutritively acceptable food supplement containing a turmeric extract or a sesquiterpenoid-based compound isolated therefrom. The health functional food of the present invention includes the form of tablets, capsules, pills or liquids, and the food to which the turmeric extract of the present invention or the sesquiterpenoid-based compound isolated therefrom can be added, for example, For example, there are various foods, beverages, gum, tea, vitamin complexes, health functional foods, and the like. In detail, the present invention provides a health functional food for improving influenza comprising a nutritively acceptable food supplement containing a turmeric extract or a sesquiterpenoid-based compound isolated therefrom.

In addition, the present invention provides isoproculcumenol (compound 1), intermedin B (compound 2), curcumadione (compound 3), (-)-curcumenone (compound 4) and ( It relates to an animal drug for preventing or treating swine influenza containing at least one compound selected from the group consisting of +)-talmeronol A (Compound 5) as an active ingredient.

In addition, the present invention provides isoproculcumenol (Compound 1), Intermedin B (Compound 2), Curcumadione (Compound 3), (-)-Curcumenone (Compound 4) and (+)-hair of the following Formula 1 Curcuma containing at least one compound selected from the group consisting of Meronol A (Compound 5) longa ) relates to an animal feed additive for improving swine influenza containing an extract as an active ingredient.

The turmeric extract of the present invention or the sesquiterpenoid-based compound isolated therefrom is added to the animal feed in an amount of 0.001 wt% to 30 wt%, preferably 0.001 wt% to 10 wt%, most preferably 0.001 wt% to 5 wt% % by weight.

In addition, the present invention provides isoproculcumenol (Compound 1), Intermedin B (Compound 2), Curcumadione (Compound 3), (-)-Curcumenone (Compound 4) and (+)-hair of the following Formula 1 Curcuma comprising at least one compound selected from the group consisting of Meronol A (Compound 5) longa ) extract as an active ingredient and relates to a natural disinfectant for preventing swine influenza.

The natural disinfectant is a composition for disinfection of influenza virus, and can be prepared in various forms such as liquid, tablet, granule, and powder.

The present invention is turmeric ( Curcuma longa ) It relates to a composition for preventing or treating swine influenza comprising an extract or a sesquiterpenoid-based compound isolated therefrom as an active ingredient, and isoprocal, a turmeric extract or a sesquiterpenoid-based compound isolated therefrom Cumenol (Compound 1), Intermedin B (Compound 2), Curcumadione (Compound 3), (-)-Curcumenone (Compound 4) and (+)-Turmeronol A (Compound 5) compounds are swine influenza It was confirmed that it had antiviral activity against the virus. Through this, the turmeric extract of the present invention or isoproculcumenol (compound 1), intermedin B (compound 2), curcumadione (compound 3), (-)-curcumenone (compound 4) and ( +)-Turmeronol A (Compound 5) compound is expected to be useful in the development of pharmaceuticals for the prevention or treatment of influenza, health functional foods, animal feed additives, or natural disinfectants.

1 is a 70% (v/v) ethanol extract of turmeric, an ethyl acetate solvent-extracted fraction of a 70% (v/v) ethanol extract, and a water-solvent-extracted fraction of 70% (v/v) ethanol extract in MDCK cells It is a graph showing cytotoxicity.
2 is a 70% (v/v) ethanol extract of turmeric, an ethyl acetate solvent-extracted fraction of a 70% (v/v) ethanol extract, and a water-solvent-extracted fraction of a 70% (v/v) ethanol extract in MDCK cells. It is a graph showing the recovery of cell lesions caused by the H1N1 influenza virus.
3 is a graph showing the cytotoxicity in MDCK cells of each small fraction of the ethyl acetate fraction of turmeric.
Figure 4 is a graph showing that each sub-fraction of the ethyl acetate fraction of turmeric recovered the cell lesion caused by the H1N1 influenza virus in MDCK cells.
5 is a thin layer chromatography pattern of each subfraction when the ethyl acetate fraction of the extract of turmeric was divided into 8 subfractions using normal phase column chromatography. From the far left, it was confirmed through UV 254 nm, UV 365 nm, and sulfuric acid flare.
6 is a chromatograph comparing the main components of F5 and F7 representatively among the eight small fractions of the ethyl acetate fraction of turmeric.

The present inventors group was studying the effect of turmeric ( Curcuma longa ) extract on swine influenza infection, observing that high antiviral activity appeared in an extract having a low content of curcumin, a representative extract component of turmeric, The present invention was completed by isolating a sesquiterpenoid-based material with high antiviral activity from the extract of

Hereinafter, preferred embodiments 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 so that this disclosure will be thorough and complete, and will fully convey the spirit of the invention to those skilled in the art.

<Example 1. Preparation of extracts and fractions of turmeric>

As a prior experiment for a small amount of turmeric prior to mass extraction for active material separation, a small amount of ethanol extract of turmeric was taken and dried using speed vacuum, and then suspended in 500 μl of distilled water. At this time, ethanol was extracted using 40-90% ethanol, and there was no significant difference in the extraction rate, but the extract using 70% ethanol with the best extraction rate (mg/ml) was used for subsequent experiments. The ethanol extract of turmeric suspended in the distilled water was solvent-extracted in the order of hexane, ethyl acetate and butanol to obtain each fraction (500 μl × 3 times). Among them, a small amount of an ethanol extract of turmeric, an ethyl acetate fraction and a water fraction of the ethanol extract were taken, dried, and dissolved in DMSO (dimethyl sulfoxide) to prepare a stock solution of 50 mg/ml.

<Example 2. Confirmation of cytotoxicity of turmeric extracts and fractions on MDCK cells>

Before performing a cell lesion effect recovery assay for the ethanol extract of turmeric, the ethyl acetate fraction and the water fraction of turmeric prepared in Example 1, to determine the concentration of the treated material that does not show toxicity to cells A cytotoxicity test was performed. After dispensing 5×10 ³ MDCK (Madin-Darby, canine kidney) cells per well in a 96-well plate, 10% FBS (fetal bovine serum), 1% penicillin/streptomycin And 0.4% L-glutamine (L-glutamine) containing DMEM (Dulbeco's Modified Eagle's Medium) medium and cultured for one day, the culture medium was discarded and washed with PBS (phosphate buffer saline). Thereafter, 100 μl of infection medium (infection media, DMEM + 0.25% bovine serum albumin (BSA) + 1 μg/ml trypsin) was added to the cells to infect for 2 hours, and then washed with PBS solution. After inoculating the swine influenza virus provided from the Central Vaccine Research Institute to a concentration of 50TCID50/100 μl/well, the final concentration of the turmeric extract and fractions prepared in Example 1 is 0.2, 1.0 μg/ml After treatment Cultured for 1 day. At this time, cells that were not treated with anything were used as normal controls.

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed using cells cultured for 1 day. MTT solution was added to the cultured cells and reacted at 37 °C, the culture medium was removed, and 100 μl of DMSO was added. After mixing well and leaving it for 10 minutes, the absorbance was measured at 570 nm to check the cell viability. At this time, the cell viability by the extracts according to each solvent was calculated based on 100% of the cell viability of the normal control group, and the results are shown in FIG. 1 . Since the level of apoptosis due to the toxicity of the turmeric extract was evaluated, the cell viability by the extract was used in the same concept as the cytotoxicity. As shown in FIG. 1, since it did not show cytotoxicity at the corresponding concentration, 0.2 and 1.0 μg/ml were used as the application concentration of the extract in the cytopathic effect recovery assay.

<Example 3. Confirmation of antiviral activity of turmeric extract and fractions>

Swine influenza virus H1N1 (A/Sw/Kor/ CAN1/04, KCTC11165BP) was used, and a cytopathic effect reduction assay (CPE reduction assay) was performed to confirm antiviral activity by analyzing the viability of cells caused by virus infection.

After dispensing 5×10 ³ MDCK cells per well in a 96-well plate, add DMEM medium containing 10% FBS, 1% penicillin/streptomycin and 0.4% L-glutamine, and incubate for one day. , the culture medium was discarded and washed with PBS. Thereafter, 100 μl of an infection medium (infection media, DMEM + 0.25% BSA + 1 μg/ml trypsin) was added to the cells to infect for 2 hours, and then washed with PBS solution. The swine influenza virus was inoculated to a concentration of 50TCID50/100 μl/well, and the final concentrations of the turmeric extract and fraction prepared in Example 1 were 0.2 and 1.0 μg/ml, and then cultured for 2 days. At this time, a cell untreated was used as a normal control, a virus treated only as a negative control, and an antiviral agent treated with 10 μM of ribavirin as a positive control.

MTT assay was performed using cells cultured for 2 days. MTT solution was added to the cultured cells and reacted at 37 °C, the culture medium was removed, and 100 μl of DMSO was added. After mixing well and leaving it for 10 minutes, the absorbance was measured at 570 nm to check the cell viability. At this time, the cell viability by the extract according to each solvent was calculated based on 100% of the cell viability of the normal control group, and the results are shown in FIG. 2 . Since the antiviral activity of turmeric extract and fractions inhibits apoptosis by influenza virus, the cell viability by the extract was used in the same concept as the antiviral activity.

As shown in FIG. 2, both the 70% ethanol extract of turmeric, the ethyl acetate fraction and the water fraction of the 70% ethanol extract at a concentration of 1 μg/ml exhibited antiviral activity. The solvent-extracted 70% ethanol-ethyl acetate fraction was the best. In particular, at 0.2 μg/ml, the fraction extracted with 70% ethanol and solvent-extracted with ethyl acetate (70% EtOH-EA) showed the strongest efficacy, whereas the water fraction showed no activity compared to the negative control. Taken together, it was found that the fraction extracted with 70% ethanol and solvent-extracted with ethyl acetate contained antiviral substances. Concentration and separation could be expected.

<Example 4. Preparation of small fractions of turmeric and confirmation of antiviral activity of small fractions>

Ethyl acetate of the extract extracted with 70% ethanol in a previous experiment with a small amount of extract Since the fraction showed the strongest antiviral activity, it was judged that the fraction contained an active substance, and sub-fractionation and material separation were carried out for a large amount of extract. After grinding 390 g of turmeric, 2 ℓ of 70% (v/v) ethanol was added, followed by ultrasonic extraction three times during one day, and concentrated under reduced pressure to obtain 83.8 g of 70% ethanol extract. The obtained extract was suspended in distilled water (1.5 L) to make a suspension. The suspension was solvent-extracted with ethyl acetate to obtain 17.2 g of an ethyl acetate fraction.

Then, the ethyl acetate fraction (17.2 g) was subjected to normal phase column chromatography (particle size: 63-200 μm) under the condition of a gradient of n -hexane/acetone (30:1 → 1:2 [v:v]) to 8 Dog small fractions (F1-8) were obtained, and MTT assay and cytopathic effect recovery assay were performed for each small fraction in the same manner as in Examples 2 and 3, and the results are shown in FIGS. 3 and 4 .

As shown in FIG. 3, no significant cytotoxicity was observed at concentrations of 0.2 and 1.0 μg/ml, and as shown in FIG. 4, small fractions F4 to F8 at concentrations of 0.2 and 1.0 μg/ml showed relatively strong activity. indicated.

At the same time, thin layer chromatography (TLC) analysis was performed to separate the active material. To this end, 8 types of small fractions were added dropwise to a plate of a normal phase together with curcumin as a standard, and developed in a solvent composition of n-hexane:acetone = 3:1. Thereafter, the polarity pattern of each material was confirmed through UV 254 nm, 365 nm and sulfuric acid flare, and the results are shown in FIG. 5 .

As shown in FIG. 5 , the small fractions F1 to F3 were the most non-polar substances, and since the properties of the small fractions were also oil, it could be expected that most of the low molecular weight essential oil components constitute the small fractions F1 to F3. In addition, curcumin, which is known to have pharmacological activity, started to elute from the small fraction F6, and it was found that it was mainly contained in F7 and F8.

In contrast, the fractions of F4 to F5, which showed antiviral activity of about F7 in FIG. 4, are non-polar than F7 and F8, in which most of the phenolic compounds, curcuminoids (curcuminoids, derivatives thereof including curcumin) occupy most, are non-polar, F1 to It is a material having a higher polarity than the essential oil component, which accounts for most of F3, and was expected to contain a compound having antiviral activity. Therefore, in subsequent experiments, the compounds included in the F5 fraction were separated and analyzed.

LC/MS analysis was performed to confirm the correct constituents. For LC/MS analysis, high performance liquid chromatography (HPLC) was performed using an Agilent 1260 Infinity system (Agilent Technologies, Inc., Santa clara, CA, USA), and the column was INNO C18 (5 μm, 4.6×250.0). mm), and finally, as a mass detector, an Agilent 6130 Quadrupole mass spectrometer was used. Analytical conditions, the mobile phase of liquid chromatography is distilled water containing 0.1% [v/v] formic acid (mobile phase A) and acetonitrile containing 0.1% [v/v] formic acid (mobile phase B), flow rate 0.6 ml It was flowed at /min, and each sample was made at a concentration of 1 mg/ml using acetone and injected by 10 μl. The conditions of the mobile phase are as follows. After saturation with 10% mobile phase B for 3 minutes before analysis, it was flowed with a gradient of 10 to 90% mobile phase B for 3.0 to 43.0 minutes, 100% mobile phase B for 43.1 to 50.0 minutes, and 10% mobile phase B for 50.1 to 55.0 minutes. Among them, the comparison results of the chromatograms of F5 and F7 are shown in FIG. 6 .

As predicted from the results of thin layer chromatography instilled together with a standard material through FIG. 6, the main compound of the small fraction F7 was curcumin, whereas the small fraction F5 contained a large number of sesquiterpenoid compounds. and UV pattern. Therefore, it was confirmed that the sesquiterpenoid compound contained in the extract isolated from turmeric exhibits antiviral activity together with curcumin.

In consideration of the high yield (1.5 g) and the antiviral activity of FIG. 4, an experiment was performed for isolating the active compound from F5.

<Example 5. Separation of sesquiterpenoid compounds>

After grinding 390 g of Korean turmeric, 2 ℓ of 70% (v/v) ethanol was added, followed by ultrasonic extraction three times a day, and concentrated under reduced pressure to obtain 83.8 g of a 70% ethanol extract. The obtained extract was suspended in distilled water (1.5 L) to make a suspension. The suspension was solvent-extracted with ethyl acetate to obtain 17.2 g of an ethyl acetate fraction. Then, the ethyl acetate fraction (17.2 g) was subjected to normal phase column chromatography (particle size: 63-200 μm ) under the condition of a gradient of n -hexane:acetone (30:1 → 1:2 [v:v]). Eight small fractions (F1-8) were obtained according to the TLC pattern.

The small fraction F5 (1.5 g) was subjected to reverse phase column chromatography (63 - 200 μm particle size) under isocratic conditions of 60% MeOH. High performance liquid chromatography on the small fraction F5.2 obtained by doing this under isocratic conditions (ACN in H ₂ O, 0-85 min: 35%, ACN containing 0.1% formic acid, H ₂ O containing 0.1% formic acid) Using high performance liquid chromatography (HPLC) (column type: Luna 5 micro Phenyl-Hexyl, column size: 250×10.0 mm, particle size: 5 μm, flow rate: 2 mL/min, UV detection: 254 nm) Compound 3 (1.1 mg, t _R = 59.4 min) was obtained. Direct HPLC for small fraction F5 (1.5 g) (ACN in H ₂ O, 0-120 min: 36%, flow rate 2 mL/min, ACN containing 0.1% formic acid, H ₂ O containing 0.1% formic acid) to compound 1 (2.8 mg, t _R = 27.3 min), compound 2 (3.7 mg, t _R = 48.7 min), compound 4 (2.0 mg, t _R = 68.5 min) and compound 5 (4.1 mg, t _R = 90.3 min) was obtained.

Based on the results of FIG. 6 , when the amounts of five kinds of sesquiterpenoid-based compounds isolated from the small fraction F5 were confirmed, the sesquiterpenoids in the fraction extracted with 70% ethanol and solvent-extracted with ethyl acetate were It was confirmed that the system compound was contained in a high concentration of 50% or more (less than 99%).

<Example 6. Confirmation of the physical and chemical structure of the compound isolated from turmeric>

Example 6-1. Isoproculcumenol (Compound 1)

Isoprocurcumenol;

brown resin;

UV (methanol) λ _max nm (log ε ) 254 (2.41);

ESIMS m/z 217.2 [M - H ₂ O + H] ⁺ , 257.2 [M + Na] ⁺ ;

¹ H-NMR (CDCl ₃ , 600 MHz): δ _H 3.22 (1H, d, J = 14.5 Hz, H-1), 1.25 (2H, m, H-2), 1.44 (2H, m, H-3 ), 1.41 (1H, s, H-5), 2.82 (2H, dd, J = 14.5, 1.3 Hz, H-6), 2.20 (1H, br s, H-9), 1.92 (3H, s, H -12), 1.82 (3H, H-13), 1.24 (1H, s, H-14), 4.91 (1H, m, H-15a), 4.90 (1H, m, H-15b).

¹³ C NMR (CDCl ₃ , 150 MHz): δ _C 51.1 (C-1), 24.7 (C-2), 28.1 (C-3), 79.8 (C-4), 58.8 (C-5), 39.7 ( C-6), 134.4 (C-7), 203.3 (C-8), 53.7 (C-9), 141.7 (C-10), 143.8 (C-11), 21.8 (C-12), 22.8 (C) -13), 24.3 (C-14), 111.5 (C-15).

Example 6-2. Intermedin B (Compound 2)

Intermedin B;

brown resin;

UV (methanol) λ _max nm (log ε ) 234 (3.15);

ESIMS m/z 217.2 [M - H ₂ O + H] ⁺ , 257.2 [M + Na] ⁺ ;

¹ H-NMR (CDCl ₃ , 600 MHz): δ _H 6.15 (1H, d, J = 10.0, 2.3 Hz, H-2), 5.77 (1H, d, J = 10.0 , H-3), 4.43 (1H , br s, H-5), 6.06 (1H, s, H-10), 1.88 (3H, s, H-12), 2.14 (3H, s, H-13), 0.90 (3H, d, J = 6.0, H-14), 4.97 (1H, s, H-15a), 5.07 (1H, s, H-15b).

¹³ C NMR (CDCl ₃ , 150 MHz): δ _C 32.9 (C-1), 133.9 (C-2), 127.2 (C-3), 145.0 (C-4), 69.5 (C-5), 31.8 ( C-6), 35.9 (C-7), 48.7 (C-8), 200.8 (C-9), 124.2 (C-10), 155.5 (C-11), 20.9 (C-12), 27.9 (C) -13), 16.8 (C-14), 113.8 (C-15).

Example 6-3. Curcumadione (Compound 3)

Curcumadione;

brown resin;

UV (methanol) λ _max nm (log ε ) 250 (2.23);

ESIMS m/z 235.2 [M + H] ⁺ , 257.2 [M + Na] ⁺ ;

¹ H-NMR (CDCl ₃ , 600 MHz): δ _H 3.07 (1H, dd, J = 16.7, 7.1 Hz, H-2a), 2.51 (1H, dd, J = 16.7, 4.7 Hz, H-2b), 2.50-2.60 (2H, m, H-3), 5.50 (1H, t, J = 6.2, H-5), 2.24 (2H, m, H-6), 2.93 (1H, dd, J = 16.7, 7.1) , H-9a), 2.59 (1H, m, H-9b), 2.45 (1H, m, H-10), 1.79 (3H, s, H-12), 1.99 (3H, s, H-13), 2.14 (3H, s, H-14), 1.07 (3H, d, J = 7.0, H-15).

¹³ C NMR (CDCl ₃ , 150 MHz): δ _C 140.9 (C-1), 28.0 (C-2), 42.9 (C-3), 208.5 (C-4), 121.4 (C-5), 30.4 ( C-6), 134.9 (C-7), 205.5 (C-8), 48.8 (C-9), 35.2 (C-10), 143.9 (C-11), 22.5 (C-12), 22.8 (C -13), 30.2 (C-14), 19.3 (C-15)

Example 6-4. (-)-curcumenone (compound 4)

(-)-Curcumenone;

brown resin;

UV (methanol) λ _max nm (log ε ) 256 (3.03);

ESIMS m/z 235.2 [M + H] ⁺ , 257.2 [M + Na] ⁺ ;

¹ H-NMR (CDCl ₃ , 600 MHz): δ _H 0.34 (1H, td, J = 7.2, 4.7 Hz, H-1), 1.49 (2H, m, H-2), 2.45 (2H, t, J ) = 6.9 Hz, H-3), 0.67 (1H, q, J = 3.9, H-5), 2.76 (2H, m, H-6), 2.54 (1H, d, J = 15.3, H-9a), 2.35 (1H, d, J = 15.3, H-9b), 2.00 (3H, s, H-12), 1.76 (3H, s, H-13), 2.06 (3H, s, H-14), 1.06 ( 3H, s, H-15).

¹³ C NMR (CDCl ₃ , 150 MHz): δ _C 23.3 (C-1), 23.0 (C-2), 43.0 (C-3), 208.3 (C-4), 23.9 (C-5), 27.5 ( C-6), 128.0 (C-7), 200.4 (C-8), 48.5 (C-9), 19.5 (C-10), 145.9 (C-11), 23.0 (C-12), 22.8 (C) -13), 29.8 (C-14), 18.7 (C-15)

Example 6-5. (+)-Turmeronol A (Compound 5)

(+)-Turmeronol A;

brown resin;

UV (methanol) λ _max nm (log ε ) 224 (2.47), 282 (1.34);

ESIMS m/z 232.2 [M + H] ⁺ , 255.1 [M + Na] ⁺ ;

¹ H-NMR (CDCl ₃ , 600 MHz): δ _H 6.64 (1H, d, J = 1.9 Hz, H-3), 6.70 (1H, dd, J = 7.9, 1.9 Hz, H-5), 7.01 ( 1H, d, H-6), 2.19 (3H, s, H-7), 3.23 (1H, m, H-8), 1.22 (3H, d, J = 6.9, H-9), 2.69 (1H, dd, J = 15.6, 6.4 Hz, H-10a), 2.58 (1H, dd, J = 15.8, 8.1 Hz, H-10b), 6.01 (1H, t, J = 1.4, H-12), 2.10 (3H) , br s, H-14), 1.85 (3H, s, H-15).

¹³ C NMR (CDCl ₃ , 150 MHz): δ _C 121.4 (C-1), 155.4 (C-2), 113.6 (C-3), 146.3 (C-4), 119.1 (C-5), 131.1 ( C-6), 15.5 (C-7), 35.4 (C-8), 22.1 (C-9), 52.7 (C-10), 200.0 (C-11), 124.2 (C-12), 153.9 (C) -13), 27.8 (C-14), 20.9 (C-15).

Therefore, five types of sesquiterpenoid compounds having antiviral activity were identified from 40-90% (v/v) ethanol-ethyl acetate fractions of turmeric, and the sesquiterpenoid compounds were isoprocal Cumenol (Compound 1), Intermedin B (Compound 2), Curcumadione (Compound 3), (-)-Curcumenone (Compound 4) and (+)-Turmeronol A (Compound 5).

<Example 7. Antiviral activity of sesquiterpenoid compounds isolated from turmeric extract (EC ₅₀ ) OK>

Antiviral activity of compounds 1 to 5 and curcumin isolated in Example 5 was confirmed. At this time, the concentration of each compound and curcumin was 2.5 μM to 500 μM, and the group not treated with the virus and the compound as a normal control group, the group treated with only the virus as a negative control group, and the antiviral agent ribavirin The group treated with was used as a positive control. The results are shown in Table 1.

delete

Compound EC ₅₀ ( μM ) One 39.10 ± 6.52 2 9.51 ± 0.54 3 40.51 ± 8.43 4 24.57 ± 4.30 5 27.60 ± 7.20 Curcumin 11.16 ± 1.97 Ribavirin 5.17 ± 0.61

As shown in Table 1 above, the EC ₅₀ (effective concentration) value of ribavirin, an antiviral agent, is 5.17 μM , and the EC ₅₀ value of curcumin is 11.16 μM for MDCK cells, indicating antiviral activity. It was confirmed that the five sesquiterpenoid compounds of the present invention exhibit antiviral activity. In particular, compound 2 exhibited superior antiviral activity even at a lower concentration than curcumin (EC ₅₀ = 9.51 μM ), and exhibited an activity close to that of ribavirin, an antiviral agent. Compounds 1, 3, 4, and 5 other than those exhibited higher EC ₅₀ values than curcumin, but were also confirmed to have antiviral activity.

<Example 8. Cytotoxicity of sesquiterpenoid compounds isolated from turmeric extract (CC ₅₀ ) OK>

In order to confirm the cytotoxicity by the compounds 1 to 5 isolated in Example 5, an MTT assay was performed. After dispensing 1×104 MDCK cells per well in a 96-well plate, DMEM medium containing 10% FBS, 1% penicillin/streptomycin, and 0.4% L-glutamine was added and cultured for one day. was discarded and washed with PBS solution. Thereafter, a new medium was added to the cells, and the compounds 1 to 5 isolated in Example 5 were treated to be 2.5 μM to 160 μM, and then cultured for 3 days. At this time, a cell untreated was used as a normal control, and curcumin, a representative indicator compound of turmeric extract, and ribavirin, an antiviral agent, were treated as positive controls 1 and 2. MTT assay was performed using cells cultured for 3 days. MTT solution was added to the cultured cells, and after reaction at 37 °C, the culture solution was removed and 200 μl of DMSO was added. After mixing well by pipetting, and leaving it for 10 minutes, the absorbance was measured at 550 nm to check the cell viability. At this time, the cell viability by each compound compared to the cell viability of the normal control was converted into a percentage (%). The results of confirming the cytotoxicity of each compound are shown in Table 2. Table 2 shows the CC ₅₀ (the half maximal cytotoxic concentration) values of the compounds against swine influenza virus.

Compound CC ₅₀ ( μM ) One > 500 2 > 500 3 > 500 4 > 500 5 > 500 Curcumin 42.06 ± 8.58 Ribavirin > 100

As shown in Table 2, in the case of a CC _{50 value indicating a cytotoxic response of 50%, the CC 50 value} of ribavirin, an antiviral agent, against MDCK cells is 100 μM or more, and the CC ₅₀ value of curcumin is 42.06 On the other hand, compounds 1 to 5 of the present invention were all 500 μM or more, and it was confirmed that there was no cytotoxicity even at a concentration higher than that of curcumin.

As can be seen from the above results, it was confirmed that compounds 1 to 5 of the present invention can be used as safe antiviral agents even at high concentrations.

<Formulation Example 1. Pharmaceutical formulation>

Formulation Example 1-1. manufacture of tablets

20 g of turmeric extract of the present invention or 200 mg of Intermedin B (Compound 2) (or isoproculcumenol (Compound 1), Curcumadion (Compound 3), (-)-Curcumenone (Compound 4) and (+)-talmeronol A (compound 5) at least one selected from the group consisting of 200 mg) were mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid, respectively. After adding a 10% gelatin solution to this mixture, it was ground and passed through a 14 mesh sieve. This was dried, and 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate were added thereto, and the resulting mixture was made into tablets.

Formulation Example 1-2. Preparation of injectable solution

Intermedin B (Compound 2) of the present invention 100 mg (or isoproculcumenol (Compound 1), Curcumadione (Compound 3), (-)-Curcumenone (Compound 4) and (+)- Termeronol A (100 mg of at least one member selected from the group consisting of compound 5), 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20 °C for 30 minutes.

<Formulation Example 2. Food Manufacturing>

Formulation Example 2-1. Preparation of cooking seasonings

Turmeric extract of the present invention or Intermedin B (Compound 2) (or isoproculcumenol (Compound 1), Curcumadion (Compound 3), (-)-Curcumenone (Compound 4) and (+) -Turmeronol A (at least one selected from the group consisting of compound 5) was added to each cooking seasoning in an amount of 1% by weight to prepare a cooking seasoning for health promotion.

Formulation Example 2-2. Flour food manufacturing

Turmeric extract of the present invention or Intermedin B (Compound 2) (or isoproculcumenol (Compound 1), Curcumadion (Compound 3), (-)-Curcumenone (Compound 4) and (+) -Turmeronol A (at least one selected from the group consisting of compound 5) is added in an amount of 0.1% by weight to flour, respectively, and the mixture is used to prepare bread, cakes, cookies, crackers and noodles for health promotion Food was prepared.

Formulation Example 2-3. Preparation of soups and gravies

Turmeric extract of the present invention or Intermedin B (Compound 2) (or isoproculcumenol (Compound 1), Curcumadion (Compound 3), (-)-Curcumenone (Compound 4) and (+) -Turmeronol A (at least one selected from the group consisting of compound 5) was added to the broth in an amount of 0.1% by weight, respectively, to prepare a health-improving soup and broth.

Formulation Example 2-4. Manufacture of dairy products

Turmeric extract of the present invention or Intermedin B (Compound 2) (or isoproculcumenol (Compound 1), Curcumadion (Compound 3), (-)-Curcumenone (Compound 4) and (+) -Tulmeronol A (at least one selected from the group consisting of compound 5) was added in an amount of 0.1% by weight, respectively, to milk, and various dairy products such as butter and ice cream were prepared using the milk.

Formulation Example 2-5. vegetable juice production

Turmeric extract of the present invention or Intermedin B (Compound 2) (or isoproculcumenol (Compound 1), Curcumadion (Compound 3), (-)-Curcumenone (Compound 4) and (+) -Turmeronol A (at least one selected from the group consisting of compound 5) was added to 1,000 ml of tomato juice or sugar root juice, respectively, to prepare vegetable juice for health promotion.

Formulation Example 2-6. fruit juice production

Turmeric extract of the present invention or Intermedin B (Compound 2) (or isoproculcumenol (Compound 1), Curcumadion (Compound 3), (-)-Curcumenone (Compound 4) and (+) -Tulmeronol A (at least one selected from the group consisting of compound 5) was added to 0.1 g each of apple juice or 1,000 ml of grape juice to prepare fruit juice for health promotion.

Claims (21)

A composition for preventing or treating swine influenza virus infection comprising a fraction obtained by suspending a 40-90% (v/v) ethanol extract concentrate of turmeric in water and then extracting it with ethyl acetate. Isoproculcumenol (Compound 1), Intermedin B (Compound 2), Curcumadione (Compound 3), (-)-Curcumenone (Compound 4) and (+)-Turmeronol of the following formula (1) without being included A composition for preventing or treating swine influenza virus infection, characterized in that 50 to 99% (w/w) of the sesquiterpenoid compound containing A (Compound 5) is contained in the ethanol-ethyl acetate extract fraction.
[Formula 1]

delete delete delete It is a health functional food for improving symptoms of swine influenza virus including a fraction obtained by suspending 40-90% (v/v) ethanol extract concentrate of turmeric in water and then extracting it with ethyl acetate. Curcumin is included as an effective active ingredient Isoproculcumenol (Compound 1), Intermedin B (Compound 2), Curcumadione (Compound 3), (-)-Curcumenone (Compound 4) and (+)-Turmeronol A of the following formula (1) A health functional food for improving symptoms of swine influenza virus, characterized in that 50 to 99% (w/w) of the sesquiterpenoid-based compound containing (Compound 5) is contained in the ethanol-ethyl acetate extract fraction.
[Formula 1]

delete delete delete An animal drug for preventing or treating swine influenza virus infection, comprising a fraction obtained by suspending a 40-90% (v/v) ethanol extract concentrate of turmeric in water and then extracting it with ethyl acetate. Isoproculcumenol (Compound 1), Intermedin B (Compound 2), Curcumadione (Compound 3), (-)-Curcumenone (Compound 4) and (+)-Tulmero of Formula 1 Animal for the prevention or treatment of swine influenza virus infection, characterized in that 50 to 99% (w/w) of the sesquiterpenoid compound containing Nol A (Compound 5) is contained in the ethanol-ethyl acetate extract fraction medicine.
[Formula 1]

delete delete delete It is an animal feed additive for improving symptoms of swine influenza virus including a fraction obtained by suspending 40-90% (v/v) ethanol extract concentrate of turmeric in water and then extracting it with ethyl acetate. Isoproculcumenol (Compound 1), Intermedin B (Compound 2), Curcumadione (Compound 3), (-)-Curcumenone (Compound 4) and (+)-Turmeronol A of the following formula (1) An animal feed additive for improving symptoms of swine influenza virus, characterized in that 50 to 99% (w/w) of the sesquiterpenoid compound containing (Compound 5) is contained in the ethanol-ethyl acetate extract fraction.
[Formula 1]

delete delete delete It is a natural disinfectant for preventing swine influenza virus infection comprising a fraction obtained by suspending 40-90% (v/v) ethanol extract concentrate of turmeric in water and then extracting it with ethyl acetate. Curcumin is not included as an effective active ingredient. Isoproculcumenol (Compound 1), Intermedin B (Compound 2), Curcumadione (Compound 3), (-)-Curcumenone (Compound 4) and (+)-Turmeronol A ( A natural disinfectant for preventing swine influenza virus infection, characterized in that 50 to 99% (w/w) of the sesquiterpenoid compound containing compound 5) is contained in the ethanol-ethyl acetate extract fraction.
[Formula 1]

delete delete delete A composition for the prevention or treatment of swine influenza virus infection, characterized in that it comprises a curcumadion (Compound 3) of Formula 1 below.
[Formula 1]

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