KR102578233B1 - Composition for preventing or treating avian influenza virus infection comprising Angelica dahurica, Curcuma longa and Resina Pini extracts - Google Patents

Abstract

The present invention relates to Angelica dahurica and Curcuma . longa ) and resin (Resina Pini) extracts as an active ingredient. It can be particularly useful in the treatment and prevention of avian influenza virus infection in animals.

Description

Composition for preventing or treating avian influenza virus infection comprising Angelica dahurica, Curcuma longa and Resina Pini extract as active ingredients {Composition for preventing or treating avian influenza virus infection comprising Angelica dahurica, Curcuma longa and Resina Pini extracts}

The present invention relates to a composition for preventing, improving or treating avian influenza virus infection, comprising as active ingredients extracts of Angelica dahurica , Curcuma longa and Resina Pini, which exhibit recovery properties against avian influenza virus infection. Related to this, and more specifically, it relates to a herbal composition having a synergistic effect in preventing, improving or treating avian influenza virus infection by mixing extracts of turmeric, turmeric and pine resin in a certain ratio.

Influenza virus is an RNA virus (Orthomyxovirus) belonging to the ‘Orthomyxo’ family and is classified into types A, B, and C among five genera. Type A is a virus that is mainly found to infect humans, and compared to types B and C, infections have been confirmed in pigs, other mammals, and various wild birds. Currently, Avian influenza, Swine influenza, and Novel flu, which are causing problems worldwide, all belong to type A viruses.

There are two types of glycoproteins, hemagglutinin (HA) and neuraminidase (NA), on the surface of the influenza virus. There are 18 types of HA and 11 types of NA, so 18 × 11 = 198 types of influenza. Viruses may occur. Humans can be infected from swine influenza, avian influenza, and zoonotic influenza originating from other animals. Swine influenza is H1N1, H1N2, H3N2, avian influenza is H5N1, H7N9, H9N2, etc. can cause infection. Additionally, there have been recent cases of avian influenza viruses being transmitted directly to humans without going through a genetic recombination process.

Avian influenza (avian flu, bird flu, highly pathogenic avian influenza, HPAI) or bird flu is an infectious respiratory disease that affects birds. All avian influenza known to date belongs to one species, 'type A influenza', and the avian influenza commonly discovered recently is caused by its variant, the H5N1 virus. This virus is divided into low pathogenicity and high pathogenicity, and in the case of high pathogenicity, it can infect humans. On March 4, 2013, a person infected with the new variant H7N9 virus died in Anhui Province, Shanghai, China. This case is the first time in the world that the H7N9 virus has infected humans. In particular, in cases like H5N1 or H7N9 that infect humans, it is difficult to make a vaccine due to the nature of the vaccine manufacturing method that uses eggs in the manufacturing process. Avian flu was first reported in Italy in the early 1900s, and is believed to be capable of spreading worldwide.

Until now, attempts have been made to develop treatments for various targets based on the proliferation and pathological infection mechanisms of the influenza A virus. Currently, as a treatment for avian, swine influenza, and swine flu, it is a selective inhibitor of neuraminidase of viral origin, which was introduced in 1996. There is an oral treatment, Tamiflu (oseltamivir phosphate), which was developed and marketed in Korea in 2001, and an inhaled drug, Relenza (zanamivir), which was marketed in 2001. Recently, as a polymerase inhibitor, it was introduced in the United States and Japan in 2018. There is the oral treatment Xofluza (baloxabir), which was approved for domestic sales in 2019 and released. Nausea, vomiting, and abnormalities in the nervous or mental system have been reported as side effects of Tamiflu, which has dominated the flu treatment market for the past 20 years. In Japan, there have been 15 cases of death in pediatric patients confirmed after Tamiflu was approved. . In addition, the emergence of Tamiflu-resistant viruses has been reported, and even human-to-human transmission of Tamiflu-resistant viruses has been discovered. Therefore, the development of viral treatments for avian and swine influenza and swine flu is essential for human health and safety, especially in the fight against avian and swine influenza and swine flu, which may occur again in humans who have experienced a single pandemic. We need more.

Looking at the infection and proliferation process of a virus, after infecting a cell, the virus creates a new virus within the cell and, in order to spread to other cells, sialic acid present outside the cell is cut using neuraminidase made by the virus. something is needed Tamiflu-resistant avian influenza viruses are known to arise through three genetic mutations (Arg292Lys, Asn274Ser, His295Tyr) that change the amino acid of the Tamiflu receptor protein to which Tamiflu usually binds. Among the three genetic mutations, in the case of the Tamiflu-resistant avian influenza virus variant in which His at 295 is changed to Tyr, which is the most expressed, the hydrophobic residue of Tamiflu is mutated to prevent it from binding to the Tamiflu receptor. . This is a virus discovered with a 10-25% probability in the 2007-2008 season in the U.S. and Europe, increasing the risk of mutation for future swine flu. With the emergence of these Tamiflu-resistant viruses, continued development of new viral treatments is required.

Meanwhile, Angelica dahurica is a biennial or triennial herb distributed in Japan, Manchuria, China, and East Siberia. The rhizome contains angelical, edultin, and phellopterin, and the seeds contain imperatorin, which has a pain-relieving effect and is effective in reducing swelling. It is reported to eliminate coldness.

Umbrella family plants have been reported to have antimicrobial and antiviral activities, and in particular, in the case of white paper, the present inventors have found that compounds of the furanocoumarin series, which are major components such as oxypeucedanin, are effective against influenza A virus. It was reported that it exhibits infection-inhibiting ability (Lee et al., 2020). The results of this study showed that furanocoumarin inhibited the infection and spread of influenza virus by acting at the early stages of the virus proliferation stage and suppressing the expression of genes that synthesize neuraminidase and nucleoprotein proteins. Nucleoprotein is one of the basic proteins that make up the structure of all ribonucleoproteins (RNPs) containing the genetic information of the virus, and is indispensable for replicating each protein that makes up the virus through transcription and translation. It's a protein that doesn't work. Therefore, white furanocoumarin-based compounds inhibit the synthesis of nucleoproteins in the early stages of infection, ultimately preventing the production of proteins that will compose progeny viruses, thus suppressing infection and spread. In addition, the influenza virus causes damage through DNA fragmentation and misuses the natural death of cells (apoptosis), causing harmful reactions to the human body such as inflammation. However, the mechanism of furanocoumarin-based compounds that exert anti-influenza effects indirectly by inhibiting abnormal cell death caused by viruses was revealed and published in the paper.

Turmeric ( Curcuma longa ) is a perennial herb widely cultivated in tropical and subtropical regions from India to Southeast Asia. Turmeric is a plant that has recently been cultivated in the southern region of Korea and is used as an ingredient in curry and other products. Turmeric contains 1-5% of aromatic essential oil and about 0.3% of curcumin, a yellow pigment, and is used as an aromatic stomach laxative for hepatitis, cholangitis, cholelithiasis, and catarrhal jaundice.

Extensive research has been conducted on the antiviral activity of curcuminoids (collectively referred to as curcumin, demethoxycurcumin, and bis-demethoxycurcumin), which are major components, including turmeric extracts. It has been proven through modern pharmaceutical science that it is effective against human immunodeficiency virus (HIV), hepatitis C virus (HCV), ebola virus, and influenza virus through various mechanisms, and is used as a health functional food in the United States. It is becoming. The present inventors also found that curcuminoid, a major component of turmeric, with respect to anti-influenza virus activity, interestingly, is activated by hemagglutinin and neuramini, which are one of the virus surface glycoproteins, in the early and final stages, respectively. It was reported that it exhibits anti-influenza activity by inhibiting neuraminidase (Chen et al., 2010; Dao et al., 2012). Hemagglutinin binds to sialic acid on the surface of host cells and helps virus particles invade cells. However, hemagglutinin is prevented from binding as the curcuminoid binds to the binding site first or causes modification of the virus envelope components, and the two compounds that are common to the three types of curcuminoid compounds It is expected that the adjacent carbonyl/enol groups and the resulting active methylene groups will contribute to activity. Meanwhile, neuraminidase replicates and combines within the cell and breaks the bond of hemagglutinin bound to sialic acid in the cell at the stage when the completed progeny virus exits the cell. It is a protein that allows the spread of viruses. However, curcuminoid compounds were confirmed to inhibit the action of neuraminidase enzyme by binding to it non-competitively.

Resina Pini is a pine tree ( Pinus ) that grows naturally in Korea, China, and Japan. Densiflora ) is a dried resin collected by cutting the stem, and is listed as a medicinal herb in Donguibogam. It is known to be effective in treating malignant tumors, swelling, itching, and pyemia, and is known to ease the internal organs and remove heat.

The diterpenoids that make up pine trees, together with essential oil components, are well known to modern people under the name phytoncide, and it has been reported that their components exhibit various antimicrobial activities, including viruses. The present inventors have discovered that abietane and labdane diterpenoid substances act in the early stages of influenza virus infection, inhibiting neuraminidase and hemagglutinin. It was recently reported that it exhibits an inhibitory effect by inhibiting the genetic synthesis of proteins, ultimately hindering the invasion and release of viruses (Ha et al., 2020). The present inventors also discovered that the compounds isolated from rosin inhibit the expression of nitric acid (NO) and nitric oxide synthase (inducible nitric oxide synthase (iNOS)), which are excessively produced due to viral infection, thereby inhibiting the The mechanisms contributing to preventing complications such as secondary bacterial pneumonia and otitis media were identified.

As described above, in the case of single substances, especially substances with a single mechanism, there is a risk of emergence of resistance, which is the reason for the emergence of drug-resistant viruses that are currently being experienced. At the same time, it cannot be used to prevent diseases in poultry and livestock because it poses a threat to humanity through zoonotic infections. However, herbal medicine extracts containing multiple ingredients, such as turmeric, turmeric, and pine resin, each with different anti-influenza activity mechanisms, have the advantage of reducing the risk of mutations at each stage because they act complexly at each stage of virus invasion. A synergistic effect of ingredients with different mechanisms can be expected. Therefore, the avian influenza inhibitor to be developed in this study is derived from a natural product used as a medicinal plant in traditional Korean medicine, so it can be used safely and is expected to have an immune-boosting effect. This combination strategy can also be expected to have the advantage of preventing the risk to human health caused by the emergence of powerfully resistant viruses as the extract acts non-selectively on each stage of virus replication.

Korean Patent No. 100962334 discloses an antiviral agent obtained from turmeric against avian, swine influenza, and swine flu, and Korean Patent No. 100743861 discloses a composition for the treatment and prevention of human diseases caused by viruses containing pine needle extract. It is starting. Additionally, Korean Patent No. 101782532 discloses a composition for preventing or treating avian influenza, swine influenza, or coronavirus containing white paper extract or furanocoumarin isolated therefrom. However, the synergistic effect against avian influenza virus caused by mixing the herbal medicine components of the present invention at a certain ratio has not been described.

Korean Patent No. 100962334, Antiviral agent against algae, swine influenza and swine flu obtained from turmeric, registered on June 1, 2010. Korean Patent No. 100743861. Composition for the treatment and prevention of human diseases caused by viruses containing pine needle extract, registered on July 24, 2007. Korean Patent No. 101782532, Composition for preventing or treating avian influenza, swine influenza or coronavirus containing white paper extract or furanocoumarin isolated therefrom, registered on September 21, 2017. Korean Patent No. 101021830, Antiviral agent against avian, swine influenza and swine flu obtained from Chrystocharix opercuratus, registered on March 7, 2011. Korean Patent No. 100950445, Antiviral agent against algae, swine influenza and swine flu obtained from licorice, registered on March 24, 2010.

Chen D.Y., et al., Curcumin inhibits influenza firus infection and haemagglutination activity, Food Chem., 119, 1346-1351, 2010. Dao T.T., et al., Curcuminoids from Curcuma longa and their inhibitory activities on influenza A neuraminidases, Food Chem., 134, 21-28, 2012. 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. Dong J.Y., et al., Sesquiterpenoids from Curcuma wenyujin with anti-influenza viral activities, Phytochemistry, 85, 122-128, 2013. Ha T.K.Q., et al., Antiviral activities of compounds isolated from Pinus densiflora (Pine Tree) against the Influenza A virus, Biomolecules, 10, 711, 2020. Hauge S.H., et al., Oseltamivir-resistant influenza viruses A (H1N1), Norway, 2007~2008, Emerg. Infect. Dis., 15, 155-162, 2009. Kim Y., et al., Vitamin C is an essential factor on the anti-viral immune responses through the production of interferon-α/β at the initial stage of influenza A virus (H3N2) infection, Immune Netw., 13, 70 -74, 2013. Lee B.W., et al., Antiviral activity of furanocoumarins isolated from Angelica dahurica against influenza A viruses H1N1 and H9N2, J. Ethnopharmacol., 259, 112945, 2020. 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:Wolters Kluwer Health, Lippincott Williams&Wilkins, 1691-1740, 2007. Zheng M., et al., Intranasal administration of chitosan against influenza A (H7N9) virus infection in a mouse model, Sci. Rep., 6, 28729, 2016. Zimmer S.M., et al., Historical perspective-emergence of influenza A (H1N1) viruses, N. Engl. J. Med., 361, 279-285, 2009.

The object of the present invention is Angelica dahurica , Curcuma longa ) and resin (Resina Pini) extracts as active ingredients to provide a composition for preventing, improving, or treating avian influenza virus infection.

Another object of the present invention is to provide a herbal composition that has an excellent synergistic effect in preventing, improving, or treating avian influenza virus infection by mixing extracts of turmeric, turmeric, and pine resin in a certain ratio.

Another object of the present invention is Angelica dahurica , Curcuma longa ) and resin (Resina Pini) extracts as an active ingredient to provide an animal drug for preventing or treating avian influenza virus infection.

The object of the present invention is Angelica dahurica , Curcuma longa ) and resin (Resina Pini) extracts as active ingredients to provide an animal feed additive for preventing or improving avian influenza virus infection.

The present invention relates to Angelica dahurica and Curcuma . longa ) and resin (Resina Pini) extracts as an active ingredient. It provides a composition for preventing or treating avian influenza virus infection.

The mixture of the white paper, turmeric and pine resin extracts may be 1 to 5:1 to 5:1 to 5 of the white paper, turmeric and pine resin extracts. Preferably, the ratio of white paper, turmeric and pine resin extracts may be 1 to 3:1 to 3:1 to 3, more preferably 1:1:3, 1:3:1, 3:1:1 or 1:1. :1, and even more preferably, the ratio of white paper, turmeric and pine resin extracts may be 1:1:3 or 1:1:1. However, the ratio was v:v:v, and a 10 mg/mL solution was used for turmeric and white paper, and a 1 mg/mL solution for pine resin was used for mixing.

The white paper extract contains furanocoumarins such as oxypeucedanin, imperatorin, phellopterin, byakangelicin, byakangelicol, and neobi. It may contain one or more selected from the group consisting of neobyakangelicol, bergapten, bergaptol, nodakenin, psoralen, and xanthotoxin, and is preferred. It may include one or more selected from the group consisting of oxypeucedanin, isoimperatorin, imperatorin, and oxypeucedanin hydrate.

The white paper extract may be obtained by extracting white paper ( Angelica dahurica ) with any one solvent selected from the group consisting of water, C1 to C4 lower alcohols, hexane, methylene chloride, ethyl acetate, and mixed solvents thereof. Preferably, the white paper extract may be an extract obtained by extracting white paper with a 50-100% C1 to C4 lower alcohol aqueous solution or lower alcohol, or a butanol fraction thereof. The white paper extract can be prepared by extracting white paper three times with an ultrasonic extractor for 2 hours using 100% ethanol, and then concentrating the fractions fractionated with butanol under reduced pressure.

The compounds included in the white paper extract can be obtained by separating the butanol fraction of the extract extracted from the white paper with a 50-100% C1 to C4 lower alcohol aqueous solution or lower alcohol, preferably the extract extracted with 100% methanol or 100% ethanol. The butanol fraction can be obtained by separating it by chromatography. The chromatography includes silica gel column chromatography, LH-20 column chromatography, RP-18 column chromatography, and thin layer chromatography. ), high performance liquid chromatography, etc. can be used.

The turmeric extract may include one or more selected from the group consisting of diarylheptanoid compounds curcumin, demethoxycurcumin, and bis-demethoxycurcumin.

The turmeric extract may be obtained by extracting turmeric with any one solvent selected from the group consisting of water, C1 to C4 lower alcohols, hexane, methylene chloride, ethyl acetate, and mixed solvents thereof. Preferably, the turmeric extract may be obtained by extracting turmeric with one or more extraction solvents selected from ethanol, methanol, acetone, ethyl acetate, or chloroform. The turmeric extract is extracted from plant components such as extraction with organic solvents (alcohol, aqueous alcohol solution, ethyl acetate, acetone, chloroform, etc.), distribution of hexane and water, use of adsorption resin such as Diaion HP-20 resin, and chromatography. It can be easily obtained by using known methods used for separation and extraction alone or in appropriate combination. The chromatography includes silica gel column chromatography, LH-20 column chromatography, thin layer chromatography (TLC), and high performance liquid chromatography. ), etc. can be used.

The pine extract is 7 α -methoxydehydroabietic acid, abieta-8,11,13,15-tetraene-18-oic acid (abieta - 8,11,13, 15-tetraen-18-oic acid), karamatsuic acid, 7-oxo-13 β - hydroxyabiet-8(14)-en-18-oic acid (7-oxo-13 β - hydroxyabiet-8(14)-en-18-oic acid), 8(14)-podocarpen-13-on-18-oic acid (8(14)-podocarpen-13-on-18-oic acid), 8 (14)-podocarpen-7,13-dione-18-oic acid (8(14)-podocarpen-7,13-dion-18-oic acid) and 4-epi-trans-communic acid (4- It may include one or more selected from the group consisting of epi-trans-communic acid).

The rosin extract may be obtained by extracting rosin with any one solvent selected from the group consisting of water, C1 to C4 lower alcohols, hexane, methylene chloride, ethyl acetate, and mixed solvents thereof. Preferably, the rosin extract is extracted from rosin (Resina Pini) with an organic solvent (alcohol, aqueous alcohol solution, ethyl acetate, ether, acetone, chloroform, etc.), distribution of hexane and water, and adsorption resin such as Diaion HP-20 resin. It can be easily obtained by using known methods used for separation and extraction of plant components, such as methods of use and methods using column chromatography, either alone or in appropriate combination. The solvent for the pine extract may be one or more extraction solvents selected from 70 to 100% ethanol or methanol. The compounds contained in the pine extract are obtained by subjecting the organic solvent extract to silica gel column chromatography, LH-20 column chromatography, thin layer chromatography (TLC), and It can be purified and separated using high performance liquid chromatography.

Meanwhile, the compounds of the present invention can be synthesized according to methods common in the art, and can also be prepared as pharmaceutically acceptable salts.

The pharmaceutical composition containing the mixture of the above-mentioned turmeric, turmeric and pine extracts is prepared in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, topical preparations, suppositories and sterile injections according to conventional methods. It can be formulated and used in the form of a solution. Carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, and cellulose. , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include the turmeric extract of the present invention or a sesquiterpenoid compound isolated therefrom and at least one excipient, e.g. For example, it is prepared by mixing starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. . Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.

The dosage of the composition will vary depending on the age, gender, and weight of the subject to be treated, the specific disease or pathological state to be treated, the severity of the disease or pathological state, the route of administration, and the judgment of the prescriber. Dosage determinations based on these factors are within the level of one skilled in the art, and dosages generally 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 can be administered to mammals such as rats, livestock, and humans through various routes. All modes of administration are contemplated, for example, oral, rectal or by intravenous, intramuscular, subcutaneous, intrauterine mucosal or intracerebrovascular injection. The turmeric extract of the present invention or the sesquiterpenoid compound isolated therefrom has little toxicity and side effects, so it is a drug that can be safely used even when taken for a long period of time for preventive purposes.

The present invention relates to Angelica dahurica and Curcuma . longa ) and Resina Pini extract as an active ingredient. A composition for preventing or treating influenza virus infection is provided.

The mixture of the white paper, turmeric and pine resin extract may be obtained by extracting the white paper turmeric and pine resin with any one solvent selected from the group consisting of water, C1 to C4 lower alcohol, hexane, methylene chloride, ethyl acetate and mixed solvents thereof. there is. The mixture of the white paper, turmeric and pine resin extracts may be a mixture of the white paper, turmeric and pine resin extracts at a weight ratio of 1 to 5:1 to 5:0.1 to 0.5. The composition for preventing or treating influenza virus infection may further include vitamin C and chitosan in the mixture of the white paper, turmeric, and pine resin extracts.

The present invention also provides white paper ( Angelica dahurica ), turmeric ( Curcuma longa ) and resin (Resina Pini) extract as an active ingredient, providing a health functional food for preventing and improving symptoms of influenza virus infection.

The present invention relates to Angelica dahurica and Curcuma . longa ) and resin (Resina Pini) extracts as an active ingredient. An animal drug for preventing or treating influenza virus infection is provided.

The present invention relates to Angelica dahurica and Curcuma . longa ) and resin (Resina Pini) extracts as an active ingredient. An animal feed additive for preventing or improving symptoms of influenza virus infection is provided.

The present invention also provides white paper ( Angelica dahurica ), turmeric ( Curcuma longa ) and resin (Resina Pini) extracts as an active ingredient. A natural disinfectant for preventing influenza virus infection is provided.

At this time, the health functional food, animal drug, animal feed additive, and natural disinfectant are a mixture of the white paper, turmeric, and pine resin extracts, respectively, water, C1 to C4 lower alcohol, hexane, methylene chloride, ethyl acetate, and these. It may be a mixture of extracts extracted with any one solvent selected from the group consisting of mixed solvents, and the mixture of the white paper, turmeric and pine resin extracts includes the white paper, turmeric and pine resin extracts in a weight ratio of 1 to 5:1 to 5:0.1 to 0.5. It may be a mixture. Additionally, the mixture of white paper, turmeric, and pine resin extracts may further include vitamin C and chitosan.

Angelica dahurica and Curcuma show cell lesion recovery ability against avian influenza virus infection according to the present invention. longa ) and Resina Pini extracts are expected to be useful as a medicine for preventing or treating avian influenza infection in animals by mixing them in a certain ratio.

Figure 1 is a graph showing the cell lesion recovery ability against H1N1 virus infection when white paper, turmeric, and pine resin extracts, including a positive control group (Ribavirin, R), were treated alone and in a 1:1:1 mixture. However, the ratio was v:v:v, and a 10 mg/mL solution was used for turmeric and white paper, and a 1 mg/mL solution for pine resin was used for mixing.
Figure 2 is a graph showing the synergistic effect of mixtures of white paper, turmeric, and pine resin extracts in various ratios on the recovery of cell lesions induced by H1N1 virus infection. However, the ratio was v:v:v, and a 10 mg/mL solution was used for turmeric and white paper, and a 1 mg/mL solution for pine resin was used for mixing.
Figure 3 is a graph showing the synergistic effect of a 1:1:1 (v:v:v) mixture of white paper, turmeric, and pine resin extracts on the recovery of cell lesions induced by H1N1 virus infection by ratio of vitamin C and chitosan mixture. However, the ratio was v:v:v, and a 1:1:1 extract mixture and a 10 mg/mL solution of vitamin C and chitosan were used for mixing.
Figure 4 is a graph showing the weight gain of mice in each group to confirm the inhibitory effect on highly pathogenic avian influenza virus (H5N2) in each group, including the group treated with the natural extract active mixture in mice. However, the concentration of the natural extract active mixture is 200 mg/mL, and the number in parentheses indicates the number of surviving mice.
Figure 5 is a graph showing the change in survival rate of mice in each group to confirm the inhibitory effect on highly pathogenic avian influenza virus (H5N2) in each group, including the group treated with the natural extract active mixture in mice. However, the concentration of the natural extract active mixture is 200 mg/mL.
Figure 6 is a graph showing the change in survival rate of chickens in each group to confirm the inhibitory effect on highly pathogenic avian influenza virus (H5N6) in each group, including the group treated with the active mixture of natural extracts in chickens. However, the concentrations of the natural extract active mixture are 50 and 10 mg/mL, respectively.
Figure 7 shows the virus titers from samples collected through swab from the upper respiratory tract of chickens in each group to confirm the inhibitory effect on highly pathogenic avian influenza virus (H5N6) in each group, including the group treated with the active mixture of natural extracts in chickens. Inoculate eggs and give EID ₅₀ This is a graph showing this method. However, the concentrations of the natural extract active mixture are 50 and 10 mg/mL, respectively.
Figure 8 shows the virus titers from samples collected through swab from the cloaca of chickens in each group to confirm the inhibitory effect on highly pathogenic avian influenza virus (H5N6) in each group, including the group treated with the active mixture of natural extracts in chickens. Inoculate at EID ₅₀ This is a graph showing this method. However, the concentrations of the natural extract active mixture are 50 and 10 mg/mL, respectively.
Figure 9 shows organs (lungs, spleen, kidneys, intestines, and brain) extracted from chickens in each group to confirm the inhibitory effect on highly pathogenic avian influenza virus (H5N6) in each group, including the group treated with the active mixture of natural extracts in chickens. Inoculate eggs with the remaining amount of virus from EID ₅₀ . This is a graph quantified using this method. However, the concentrations of the natural extract active mixture are 50 and 10 mg/mL, respectively.

While researching compounds with pharmacological activity of herbal extracts including turmeric, turmeric, and pine resin, the present inventor confirmed that the herbal extract of the present invention had an excellent antiviral effect in animals infected with avian influenza, and completed the present invention.

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, the content introduced herein is provided to be thorough and complete, and to fully convey the spirit of the present invention to those skilled in the art.

< Example 1. Blank paper, Turmeric and Songji Preparation of extract mixture>

Turmeric ( Curcuma longa ) was supplied in powder form by freeze-drying 70% alcohol extract from a Chinese company and used to prepare the mixture.

9.6 kg of white paper ( Angelica dahurica ) was extracted three times for a total of 1 hour and 30 minutes each time using an ultrasonic extractor using 70% ethanol. Afterwards, the extract was concentrated under reduced pressure, freeze-dried into powder form, and used to prepare the mixture.

Resina Pini purchased the extract from Deokhyeondang, a herbal medicine store in Jegi-dong, Seoul, and used it to prepare the mixture.

Samples 1 to 10 were prepared by dissolving the white paper, turmeric, and pine extracts obtained above in DMSO in the ratios shown in Table 1 below and mixing them (v/v). However, in the case of pine resin, preliminary experiments showed cytotoxicity when treated to 10 ㎍/mL, and strong activity at 1 ㎍/mL. Therefore, a 10 mg/mL solution was used as a standard solution for turmeric and white paper, and a 1 mg/mL solution was used as a standard solution for resin. For the convenience of the following experiments, the ratio was expressed as the volume ratio of the reference solution.

Turmeric (CL) ^* Blank paper (AD) ^* Song Ji (PD) ^* sample 1 One 0 0 sample 2 0 One 0 sample 3 0 0 One sample 4 One One One sample 5 One One 3 sample 6 One 3 One sample 7 3 One One sample 8 One One 5 sample 9 One 5 One sample 10 5 One One

^* However, the ratio is v:v:v, and a 10 mg/mL solution is used for turmeric and white paper, and a 1 mg/mL solution is used for pine resin.

< Example 2. Blank paper, Turmeric , Songji caused by influenza viruses in extracts and their mixtures. Cellular lesion Recovery effect>

The ability of samples 1 to 4 prepared in Example 1 to recover from cellular lesions caused by influenza virus (H1N1) was examined. Influenza virus H1N1 (A/Sw/Kor/CAN1/04, KCTC11165BP) provided by the Central Vaccine Research Institute was used, and the cytopathic effect recovery test was used to confirm antiviral activity by analyzing the survival rate of cells caused by virus infection. Cytopathic effect reduction assay (CPE reduction assay) was performed.

After dispensing 1 × 10 ⁴ MDCK cells per well in a 96 well plate, add DMEM medium containing 10% FBS, 1% penicillin/streptomycin, and 0.4% L-glutamine, and culture for one day. , the culture medium was discarded and washed with PBS solution. Afterwards, new medium was added to the cells, samples 1 to 4 were treated to concentrations of 0.01, 0.1, 1, and 10 μg/mL, and cultured for 3 days. At this time, cells that were not treated with anything were used as a normal control (Control), and cells treated with 10 μM ribavirin, an antiviral agent, were used as a positive control, and each experimental group was repeated three times.

MTT assay was performed using cells cultured for 3 days. MTT solution was added to the cultured cells and reacted at 37°C, then the culture medium was removed and 200 ㎕ of DMSO was added. After mixing well by pipetting and leaving for 10 minutes, the absorbance was measured at 550 nm to check the survival rate of the cells. At this time, the cell survival rate by each compound compared to the cell survival rate of the normal control group was converted into percentage (%), and the results are shown in Figure 1.

As shown in Figure 1, MDCK cells, whose cell viability was reduced to about 24% by the H1N1 virus, were killed by turmeric (CL), white paper (AD), and pine resin (PD) extracts (samples 1 to 3) at the same concentration. Survival rate increased. In particular, at a concentration of 10 μg/mL of AD extract, cell survival rate increased to about 53%, confirming good antiviral activity. Turmeric, well known for its curcumin activity, also showed antiviral activity in a concentration-dependent manner. In the case of pine resin, the antiviral activity increased to 59.3% at 1 ㎍/mL, confirming good antiviral activity, but at a higher concentration of 10 ㎍/mL, the cell viability decreased to about 20%, showing cytotoxicity, which was observed at a certain concentration. In the case of application of pine extract, it can be seen that there is a problem that side effects due to cytotoxicity must be considered. In addition, considering that rosin is non-polar in nature and has extremely low solubility, so it can only be used in small volumes at low concentrations, it was determined that it was necessary to apply a mixed composition that could safely treat rosin extract at low concentrations and increase the effect.

In the case of a substance mixed with extracts of turmeric (CL), white paper (AD), and pine resin (PD) at a standard solution volume ratio of 1:1:1 (sample 4), it showed stronger antiviral activity at the same concentration compared to a single substance. Confirmed. In particular, higher antiviral activity was confirmed at a concentration of 1 ㎍/mL of the turmeric (CL), Baekji (AD), and pine root (PD) extract mixture than the effect at a concentration of 10 ㎍/mL of the Baekji (AD) extract, demonstrating safe and high antiviral activity. was found to have.

It is believed that the synergistic antiviral effect of turmeric (CL), turmeric (AD), and pine root (PD) extracts is due to the different mechanisms of each plant. In the case of turmeric (Chen et al., 2010; Dao et al., 2012), curcuminoids, including curcumin, which is the main component, interact with hemagglutinin, the surface glycoprotein of the influenza virus. It has been reported that it inhibits viral infection and spread by inhibiting neuraminidase. In addition, in the case of white paper (Lee et al., 2020), major furanocoumarins, including oxypeucedanin, which is the main component including the extract, enter the cell and are essentially involved in making virus constituent proteins. It has been found that it inhibits influenza virus infection by inhibiting the synthesis of nucleoprotein. In the case of pine (Ha et al., 2020), it was reported that diterpenoid, a major component, inhibits the cytopathic effect by inhibiting the expression of mRNA rather than acting with the surface glycoprotein of the influenza virus. Therefore, the three different plant extracts inhibit the infection and activity of influenza viruses through different mechanisms, and the virus-suppressing effect of each plant extract creates conditions that can further demonstrate the antiviral effect of each plant extract. It can be said that it shows a synergistic effect.

< Example 3. Blank paper, Turmeric and Songji Antiviral activity of mixture by extract ratio>

In order to confirm the antiviral activity characteristics of the mixture of extracts of white paper (AD), turmeric (CL), and pine resin (PD) by ratio, samples 4 to 10 prepared in Example 1 were mixed with turmeric (CL), white paper (AD), and The cell lesion recovery ability of mixtures of pine resin (PD) extracts at concentrations of 0.01, 0.1, 1, and 10 μg/mL was confirmed.

In the same manner as Example 2, 1 × 10 ⁴ cells per well were placed in a 96 well plate. After dispensing MDCK cells and culturing them for 24 hours, the culture medium was discarded, and new medium was treated with a mixture of turmeric (CL), white apricot (AD), and pine tree (PD) extracts at 0.01, 0.1, 1, and 10 μg/mL. Then it was cultured for 3 days. At this time, cells that were not treated with anything were used as a normal control, and 10 μM ribavirin, an antiviral agent, was used as a positive control, and each experimental group was repeated three times. MTT assay was performed using cells cultured for 3 days, and the cell survival rate by each compound compared to the cell survival rate of the normal control group was converted into percentage (%) and the results are shown in Figure 2.

As shown in Figure 2, when samples 4 to 10 mixed with turmeric (CL), pine resin (PD), and algae (AD) extract standard solutions at a certain volume ratio were administered, the results were 1:1:1, 1:3:1. , 1:1:3 and 5:1:1(v:v:v) showed high virus inhibition ability, and when treated at 1 ㎍/mL, 1~5:1~5:1~5(v:v :v) showed virus suppression ability in all cases. In addition, referring to the results of the inventors' preliminary experiment, the ratios of 6~10:1:1, 1:6~10:1, and 1:1:6~10 (v:v:v) outside of this ratio, that is, single As the proportion of plants increases, the synergistic effect of the mixture of low-concentration turmeric (CL), pine tree (PD), and pine tree (AD) extracts increases with the effect of the turmeric (CL), pine tree (PD), and pine tree (AD) extracts alone, respectively. Since it can be expected to appear similar to the cytotoxic effect of :v:v) mixture (1:1:0.1 by weight) was confirmed to be the most suitable and was used in subsequent experiments.

< Example 4. Blank paper, Turmeric and Songji Confirmation of antiviral activity of extract mixture and vitamin C and chitosan mixture>

In the case of vitamin C and chitosan, according to previously reported research results, the efficacy of vitamin C and chitosan against anti-influenza viruses has been confirmed in in vivo studies (Zheng et al., 2016; Kim et al., 2013), and animal models Alternatively, when applied to the human body, a synergistic effect of medicinal efficacy can be expected by applying it together with a mixture of white paper, turmeric, and pine resin extracts of the present invention. Therefore, the antiviral activity of a 1:1:1 (v:v:v) mixture of white paper, turmeric, and pine extracts mixed with vitamin C and chitosan was confirmed. Combination ratio of 1:1:1 (v:v:v) mixture of turmeric (CL), white paper (AD) and pine resin (PD) extracts prepared in the same manner as in Example 1 and vitamin C and chitosan was prepared as shown in Table 2. However, the ratio is the standard solution volume ratio (v:v:v), and 10 mg/mL solutions of vitamin C and chitosan each were used as reference solutions.

Mixture(v:v:v)
(CL:AD:PD=1:1:1) Vitamin C chitosan sample 4 10 0 0 sample 11 9 0 One sample 12 9 One 0 sample 13 9 0.5 0.5

^* However, the ratio is v:v:v, and a 10 mg/mL solution is used for turmeric, white paper, vitamin C, and chitosan, and a 1 mg/mL solution is used for resin.

In the same manner as Example 2, 1 × 10 ⁴ cells per well were placed in a 96 well plate. After MDCK cells were dispensed and cultured for 24 hours, the culture medium was discarded and samples 11 to 14 prepared at the ratios in Table 2 were treated with 0.01, 0.1, 1, and 10 μg/mL and cultured for 3 days. At this time, cells that were not treated with anything were used as a normal control, and 10 μM ribavirin, an antiviral agent, was used as a positive control, and each experimental group was repeated three times. MTT assay was performed using cells cultured for 3 days, and the cell survival rate by each compound compared to the cell survival rate of the normal control group was converted into percentage (%) and the results are shown in Figure 3.

As shown in Figure 3, vitamin C or chitosan was added to the mixture (sample 4) of pine root (PD), turmeric (CL), and turmeric (AD) extracts mixed at 1:1:1 (v:v:v). The added mixture (samples 11 and 12) tended to have higher antiviral activity at slightly lower concentrations than the 1:1:1 mixture of turmeric, white paper, and pine resin treated alone. However, a mixture (sample 13) in which vitamin C and chitosan were added to pine tree (PD), turmeric (CL) and white paper (AD) extracts (1:1:1) was (AD) It was confirmed that it exhibited higher antiviral activity even at 1/10 the concentration than the extract or samples 11 and 12 to which only vitamin C or chitosan was added. Through this, it was confirmed that the antiviral effect of the turmeric, white paper, and pine resin mixture could be increased by adding vitamin C and/or chitosan to the white paper, turmeric, and pine resin mixture. The results of Examples 2 to 4 can be shown in Table 3 as follows.

Turmeric
(CL) blank
(AD) Rosin
(PD) Cell viability(%) 0.01(㎍/mL) 0.1(㎍/mL) 1(㎍/mL) 10(㎍/mL) Control - 100± H1N1 virus - 23.8± Ribavirin
(10 μM) - 69.8± Ribavirin 31.6% 39.8% 42.6% 68.04% sample 1 One 0 0 26.8% 30.4% 38.1% 38.8% sample 2 0 One 0 29.1% 46.4% 47.1% 53.3% sample 3 0 0 One 30.6% 50.2% 59.3% 19.9% sample 4 One One One 32.4% 50.6% 56.6% 62.7% sample 5 3 One One 23.4% 24.4% 36.6% 40.7% sample 6 One 3 One 34.1% 38.0% 57.9% 60.5% sample 7 One One 3 42.4% 47.8% 55.2% 60.6% sample 8 5 One One 48.7% 50.4% 53.4% 57.0% sample 9 One 5 One 36.2% 41.3% 43.3% 57.2% sample 10 One One 5 25.8% 35.2% 44.3% 56.0% 1:1:1 test 1:1:1 vitaC chito sample 11 9 One 0 33.0% 42.9% 52.2% 60.9% sample 12 9 0 One 35.2% 45.6% 50.7% 59.3% sample 13 9 0.5 0.5 34.4% 50.6% 58.5% 59.8%

^* However, the ratio is v:v:v, and a 10 mg/mL solution is used for turmeric and white paper, and a 1 mg/mL solution is used for pine resin.

< Example 5. Blank paper, Turmeric and Songji Confirmation of antiviral activity of extract mixture in mice infected with avian influenza>

The active mixture of natural extracts identified in Example 4 was applied to an avian influenza virus infection animal model to confirm antiviral activity. For this purpose, extracts of white paper, turmeric, and pine resin were dissolved in PEG (Polyethylene glycol):DMSO:H ₂ O mixed solution (1:8:16, v/v/v) to obtain a 1:1:0.1 (w/w/w) solution. After mixing in proportions, the prepared natural extract mixture, vitamin C, and chitosan were mixed in a ratio of 9:0.5:0.5 (v/v/v) to finally complete the natural extract active mixture. In addition, referring to previously reported papers related to this, samples were prepared at 200 mg/mL for mouse experiments, and the positive control group was prepared at 1 mg/mL in the same mixed solution using oseltamivir. Finally, a mixed solution was prepared for the untreated control group and the negative control group.

The highly pathogenic avian influenza virus mouse adapted H5N2 Ab/Korea/ma81/07 (Av/ma81) provided by Chungbuk National University was used as the avian influenza virus, and the survival rate and weight gain of mice due to virus infection were used as indicators of the inhibitory effect on avian influenza. Confirmed.

For this experiment, 36 Balb/c mice aged 6 to 8 weeks were used, 9 per group (G1, test group; G2, positive control; G3, negative control; G4, untreated control). Each group was administered 6 hours before vaccination. 200 mg/ml of the natural extract active mixture was administered at an amount of 100 ㎕/mouse using a sonde tool. And 6 hours after feeding the mixture, the animals were challenged with influenza virus. After anesthetizing the mouse by inoculating it into the abdominal ^cavity with Zoletil®50, the mouse nasal cavity was inoculated with 20 ㎕/dose of influenza virus 10 ^6.5 EID ₅₀ /ml (5LD ₅₀ , 50% lethal dose; lethal dose 50). The extract mixture was fed at the same time for 7 days, and the survival rate and weight gain for each group were checked. Three days after the challenge, blood was collected from 6 mice per group, excluding the untreated control group. The lungs of mice that survived 14 days after challenge were removed and histological analysis was performed, and the results are shown in Figures 4 and 5.

As shown in Figure 4, the body weight of the avian influenza virus-administered group (G3) decreased rapidly until the 8th day of virus inoculation compared to the untreated group (G4). However, after 8 days, the average weight of the surviving mice rapidly recovered to that of the untreated control group, which is believed to be the result of the recovery of a small number of mice that survived through a natural healing process from avian influenza virus infection. However, as shown in Figure 5, the survival rate on day 6 of infection is 33%, which is very low.

In the case of the positive control group (G2), in which mice infected with avian influenza virus were treated with Tamiflu, mouse body weight decreased compared to the untreated group (G4), but the weight loss was less than that in the negative control group (G3), in which only avian influenza virus was administered. Additionally, as shown in Figure 5, the survival rate after 6 days of infection was about 88%, showing a high survival rate.

When the mixture of white paper, turmeric and pine resin extracts of the present invention was applied to mice infected with avian influenza virus, the mouse body weight showed a decrease similar to that of the positive control group treated with Tamiflu. In addition, as shown in Figure 5, the survival rate after 6 days of infection was about 67%, which was lower than that of the positive control group, but showed a survival rate that was more than twice that of the negative control group, indicating that the mixture of white paper, turmeric, and pine resin extracts of the present invention was effective in avian influenza virus infection. It was confirmed to be effective in preventing/treating mice.

< Example 6. Blank paper, Turmeric and Songji Avian Influenza Virus Infection with Extract Mixtures in chicken Confirmation of antiviral activity>

6.1 Blank paper, Turmeric and Songji Effect of extract mixture on survival rate of chickens infected with avian influenza virus

The natural extract active mixture prepared in Example 4 was evaluated in a biosafety level 3 research facility (BL3) to determine its ability to inhibit High Pathogenic Avian Influenza virus (HPAI) in chickens. First, a composition for preventing or treating avian influenza infection was prepared in the same manner as in Example 5, and the positive control group was prepared using oseltamivir at 1 mg/mL in the same mixed solution. Finally, a mixed solution was prepared for the untreated control group and the negative control group. An experiment was conducted with 10 4-week-old SPF (specific pathogen free) chicks per group. First, 4 hours before the challenge, the SPF chickens were inoculated with a low concentration (Group 1, 10 mg/kg) or high concentration (Group 2, group 2) of the natural extract active mixture. 50 mg/kg) was mixed and fed for a total of 5 days (1dpi - 5dpi). 6 hours after the first feeding, the highly pathogenic avian influenza virus A/AP/Korea/W612/2017(H5N6) acquired from Chungbuk National University was administered to the untreated control group (200 ㎕/dose) at a concentration of 10 ^3.5 EID ₅₀ /ml (5LD50). All groups except Group 5) were challenged intranasally. The control group was treated with the same amount of solvent in the extract mixture and fed. After vaccination, the survival rate up to day 7 is shown in Table 4 and Figure 6.

Survival rate (%) experimental group 1 dpi 2dpi 3dpi 4dpi 5dpi 6dpi 7dpi G1 (low concentration experimental group) 100 83.3 50.0 50.0 33.3 33.3 33.3 G2 (high concentration experimental group) 100 83.3 83.3 83.3 83.3 83.3 83.3 G3 (positive control: Tamiflu) 100 83.3 83.3 83.3 66.7 66.7 66.7 G4 (Voice control group: Challenge) 100 66.7 66.7 33.3 16.7 0 0 G5 (untreated control group) 100 100 100 100 100 100 100

As shown in Table 4 and Figure 6, the negative control group (G4) infected with avian influenza virus died 100% on the 6th day of infection, and the positive control group (G3) treated with Tamiflu survived 66.7% on the 6th day of infection. When treated with the active mixture of natural extracts of the present invention, the low-concentration experimental group (G1) showed a survival rate of 33.3%, confirming that the survival rate increased compared to the negative control group, and in the case of the high-concentration experimental group (G2), the survival rate was 83.3%, which was 66.7%. The survival rate was higher than that of the positive control group treated with intamiflu. Therefore, it was confirmed that the active mixture of natural extracts effectively inhibits the proliferation of highly pathogenic avian influenza viruses, especially in birds, and that this action occurs in a concentration-dependent manner. Currently, in Korea, starting from November 26, 2020, highly pathogenic avian influenza (H5N8) has been confirmed at 43 farms in 6 cities and provinces nationwide, and infected poultry have been buried and culled, resulting in enormous economic loss. is experiencing difficulties. Since the avian influenza virus has a wider spread through the movement of migratory birds, which are its hosts, compared to the swine influenza virus, it occurs periodically in Korea and causes greater damage. In this situation, if it is possible to reduce the infection rate and increase the survival rate of livestock farm poultry through the infection and spread inhibition ability of the natural product complex against the highly pathogenic avian influenza virus identified in the present invention, the ripple effect in economic and environmental aspects will be It is judged to be significant.

6.2 Virus titration in organs of chickens infected with avian influenza virus

For the chickens that survived and died in Example 6.1, two birds from each test group and the control group were sacrificed on the 4th day after the virus challenge, and the lungs, spleen, kidneys, small intestine, and brain were removed. Afterwards, the virus was pulverized and diluted and the virus titer (EID50) was measured using a hemagglutination test (HA test). The antigen agglutination reaction test is a test that determines the titer of a virus by using the binding property of a virus containing the surface glycoprotein hemagglutinin when it encounters red blood cells. When positive, the virus agglutinates red blood cells and spreads widely, but when negative, the virus cannot coagulate, causing red blood cells to sink and flow when tilted. The red blood cells used were 0.5% chicken, turkey RBC (red blood cell; cRBC, tRBC), and PBS (Phosphate buffered saline) was used for dilution. Using a U bottom plate, 50 μl of PBS was added to each well of a 96 well plate. 50 ㎕ of virus was taken so that the HA titer corresponds to a dilution factor of 4-8, and the virus was dispensed one by one into the first well corresponding to a dilution factor of 2, and then step-by-step dilution was performed for the next well by 2-fold dilution. After the procedure, 50uL of RBCs were added at a time without touching the tip. Afterwards, after adding it, the plate was gently struck. As a negative control, to check the condition of RBCs, RBCs were placed in the last well containing only PBS. After incubation for 30 minutes, aggregation was checked and the virus titer (EID ₅₀ ) was measured. The results are shown in Table 5 and Figure 7.

Experimental group (EID ₅₀ /0.2ml) Lung Spleen Kidney Intestine Brain G1 (low concentration experimental group) 4.0 2.3 3.0 4.3 2.0 G2 (high concentration experimental group) 0.5 0.2 0.3 0.5 0.2 G3 (positive control: Tamiflu) 1.3 1.3 1.7 2.3 1.0 G4 (Voice control group: Challenge) 4.3 2.7 3.0 4.7 2.0 G5 (untreated control group) 0.1 0.1 0.1 0.1 0.1

As shown in Table 5 and Figure 7, the avian influenza virus infection negative control group (G4) showed high levels of virus in the lungs, spleen, kidneys, small intestine, and brain, especially in the lungs and intestines. In the case of the positive control group (G3) treated with Tamiflu, the viral load was reduced in all tested organs compared to the negative control group. In particular, the decrease was large in the lungs, but the small intestine showed a high viral load. When treated with the active mixture of natural extracts of the present invention, there was no significant difference from the negative control group in the low-concentration experimental group (G1), but in the case of the high-concentration experimental group (G2), the viral load was significantly increased in the lungs, spleen, kidneys, small intestine, and brain. It was reduced to a level lower than that of the positive control group.

6.3 Virus titration in the respiratory tract of chickens infected with avian influenza virus

Influenza virus, which is an attack strain, is a virus that occurs in birds. Since the virus generally proliferates in the respiratory tract (airway, lungs) and cloaca of birds, continuous swabbing after infection can be performed to monitor the growth curve of the virus and the results of its death. there is. Specifically, the H5 type virus, which is classified as a highly pathogenic influenza virus, spreads and proliferates not only in the general respiratory tract and cloaca but also in various organs such as the brain, spleen, and kidneys. Therefore, when inoculated with highly pathogenic influenza, tissues must be removed and the virus titer measured. It is possible to effectively monitor the pathogenicity of the virus and the degree of virus reduction by vaccines or therapeutic substances. Therefore, swab samples were obtained from the upper respiratory tract of the chickens that survived in Example 6.1 for 5 days, and the virus titer was titrated in the same manner as in Example 6.2. The results are shown in Table 6 and Figure 8.

Experimental group (EID ₅₀ /0.2ml) 1 dpi 2dpi 3dpi 4dpi 5dpi G1 (low concentration experimental group) 2.67 3.50 3.67 2.50 1.33 G2 (high concentration experimental group) 2.67 1.00 0.80 0.50 0.10 G3 (positive control: Tamiflu) 2.50 1.33 1.50 1.67 0.50 G4 (Voice control group: Challenge) 2.50 4.17 4.50 4.17 1.33 G5 (untreated control group) 0.10 0.10 0.10 0.10 0.10

As shown in Table 6 and Figure 8, the avian influenza virus infection negative control group (G4) showed a very high viral load in the respiratory tract of chickens compared to the untreated control group (G5) from the first day of infection. Afterwards, it increased on the 2nd and 3rd days, decreased slightly on the 4th day, and decreased to about half of the level on the 1st day of infection on the 5th day. However, on the 6th day of infection, 100% of the negative control group (G4) died.

The positive control group (G3) treated with Tamiflu showed a viral load similar to the negative control group (G4) on the 1st day of infection, but on the 2nd day, it decreased to half that of day 1, increased slightly on the 3rd and 4th days, and reached 0.5 EID ₅₀ on the 5th day. / decreased to 0.2ml. When treated with the active mixture of natural extracts of the present invention, the low concentration experimental group (G1) showed a viral load similar to the negative control group (G4) on the 1st day of infection, gradually increased on the 2nd and 3rd days, and decreased from the 4th day. In the case of the high-concentration experimental group (G2) of the present invention, the viral load was similar to that of the negative control group (G4) on the 1st day of infection, but the viral load in the organs decreased rapidly from the 2nd day, and further decreased on days 2-4, until 5. On day 1, almost no virus was detected, similar to the untreated control group (G5).

6.4 Virus titration in the cloaca of chickens infected with avian influenza virus

A swab sample was obtained from the cloaca of a surviving chicken, and the virus titer was titrated in the same manner as in Example 6.2, and the results are shown in Table 7 and Figure 9.

Experimental group (EID ₅₀ /0.2ml) 1 dpi 2dpi 3dpi 4dpi 5dpi G1 (low concentration experimental group) 0.50 3.17 3.50 3.00 1.67 G2 (high concentration experimental group) 0.50 1.00 0.83 0.50 0.10 G3 (positive control: Tamiflu) 0.50 1.17 1.17 1.50 0.50 G4 (Voice control group: Challenge) 0.50 3.50 4.00 4.17 1.67 G5 (untreated control group) 0.10 0.10 0.10 0.10 0.10

As shown in Table 7 and Figure 9, the avian influenza virus infection negative control group (G4) showed an increase from the second day of infection, and the amount of virus was very high in the cloaca of chickens compared to the untreated control group (G5) on the second day of infection. indicated. Afterwards, it increased on the 3rd and 4th days, and decreased on the 5th day, decreasing to about half of the level on the 1st day of infection. However, on the 6th day of infection, 100% of the negative control group (G4) died.

The positive control group (G3) treated with Tamiflu showed a viral load similar to the negative control group (G4) on the 1st day of infection, and on the 2nd day, the viral load increased more than twice that of the 1st day, but was less than half that of the negative control group (G4). Activity could be confirmed. Afterwards, it increased slightly on the 3rd and 4th days and then decreased to 0.5 EID ₅₀ /0.2ml on the 5th day. When treated with the active mixture of natural extracts of the present invention, the low concentration experimental group (G1) showed a viral load similar to the negative control group (G4) on the 1st day of infection, and the viral load was similar to the negative control group (G4) on the 2nd and 3rd days. It increased rapidly. However, it decreased from the 4th day of infection, and on the 5th day, it decreased to about half of the level on the 1st day of infection. On the 6th day of infection, 66.7% of the low concentration test group (G1) died.

In the case of the high concentration experimental group (G2) of the present invention, the viral load was similar to that of the negative control group (G4) on the first day of infection, and on the second day it increased compared to the first day, but the level of virus increase was lower than that of the positive control group (G3). Afterwards, it continued to decrease on the 3rd and 4th days, and on the 5th day of virus infection, almost no virus was detected, similar to the untreated control group (G5).

The above results suggest that the composition of the present invention, which is made up of 100% natural products with almost no side effects, can exhibit an antiviral effect equivalent to or greater than that of Tamiflu in animals infected with avian influenza virus. In particular, it has an excellent antiviral effect in poultry, including chickens. It is thought to exhibit a viral effect.

< example 1. Pharmaceutical preparations>

example 1-1. manufacture of tablets

20 g of the mixture of white paper, turmeric and pine resin extracts of the present invention, vitamin C and chitosan mixture were mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid, respectively. A 10% gelatin solution was added to this mixture, then pulverized 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.

example 1-2. Manufacturing of injectable solutions

100 mg of a mixture of white paper, turmeric and pine resin extracts of the present invention, 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.

Claims (14)

Contains a mixture of extracts of Angelica dahurica, Curcuma longa and Resina Pini as active ingredients. In the pharmaceutical composition for preventing or treating avian influenza virus infection,
A pharmaceutical composition for preventing or treating avian influenza virus infection, wherein the avian influenza virus is an H1N1, H5N2, or H5N6 influenza virus. According to paragraph 1,
The mixture of the white paper, turmeric, and pine resin extracts is a mixture of white paper turmeric and pine resin extracted with any one solvent selected from the group consisting of water, C1 to C4 lower alcohol, hexane, methylene chloride, ethyl acetate, and mixed solvents thereof. A pharmaceutical composition for preventing or treating avian influenza virus infection, characterized in that: According to paragraph 1,
The mixture of the white paper, turmeric and pine resin extracts is a pharmaceutical composition for the prevention or treatment of avian influenza virus infection, characterized in that the white paper, turmeric and resin extracts are in a weight ratio of 1 to 5:1 to 5:0.1 to 0.5. According to paragraph 1,
The white paper extract contains one or more selected from the group consisting of oxypeucedanin, isoimperatorin, imperatorin, and oxypeucedanin hydrate,
The turmeric extract includes one or more selected from the group consisting of curcumin, demethoxycurcumin, and bis-demethoxycurcumin,
The pine extract is 7 α -methoxydehydroabietic acid, abieta-8,11,13,15-tetraene-18-oic acid (abieta - 8,11,13, 15-tetraen-18-oic acid), karamatsuic acid, 7-oxo-13 β- hydroxyabiet-8(14)-en-18-oic acid (7-oxo-13 β- hydroxyabiet-8(14)-en-18-oic acid), 8(14)-podocarpen-13-on-18-oic acid (8(14)-podocarpen-13-on-18-oic acid), 8 (14)-podocarpen-7,13-dione-18-oic acid (8(14)-podocarpen-7,13-dion-18-oic acid) and 4-epi-trans-communic acid (4- A pharmaceutical composition for preventing or treating avian influenza virus infection, comprising at least one selected from the group consisting of epi-trans-communic acid. According to any one of claims 1 to 4,
A pharmaceutical composition for preventing or treating avian influenza virus infection, characterized in that it further comprises vitamin C and chitosan in the mixture of the white paper, turmeric and pine resin extracts. In the animal drug for preventing or treating avian influenza virus infection, comprising a mixture of extracts of Angelica dahurica, Curcuma longa and Resina Pini as active ingredients,
An animal drug for preventing or treating avian influenza virus infection, wherein the avian influenza virus is an H1N1, H5N2, or H5N6 influenza virus. According to clause 6,
The mixture of the white paper, turmeric, and pine resin extracts is a mixture of white paper turmeric and pine resin extracted with any one solvent selected from the group consisting of water, C1 to C4 lower alcohol, hexane, methylene chloride, ethyl acetate, and mixed solvents thereof. An animal drug for preventing or treating avian influenza virus infection, characterized in that: According to clause 6,
The mixture of the white paper, turmeric and pine resin extracts is an animal drug for the prevention or treatment of avian influenza virus infection, characterized in that the white paper, turmeric and resin extracts are in a weight ratio of 1 to 5:1 to 5:0.1 to 0.5. According to any one of claims 6 to 8,
An animal drug for preventing or treating avian influenza virus infection, characterized in that the mixture of the above-mentioned white paper, turmeric and pine resin extracts further contains vitamin C and chitosan. According to clause 9,
The animal drug is an avian influenza virus, characterized in that it targets one animal selected from the group consisting of wild birds such as chickens, turkeys, geese, ducks, pheasants, quail, pigeons, ostriches, poultry, pigs, dogs, and cats. Animal medicines for the prevention or treatment of infections. In the animal feed additive for preventing or improving symptoms of avian influenza virus infection, comprising a mixture of extracts of Angelica dahurica, Curcuma longa and Resina Pini as an active ingredient,
An animal feed additive for preventing or improving symptoms of avian influenza virus infection, wherein the avian influenza virus is an H1N1, H5N2, or H5N6 influenza virus. According to clause 11,
The mixture of the white paper, turmeric, and pine resin extracts is a mixture of white paper turmeric and pine resin extracted with any one solvent selected from the group consisting of water, C1 to C4 lower alcohol, hexane, methylene chloride, ethyl acetate, and mixed solvents thereof. An animal feed additive for preventing or improving symptoms of avian influenza virus infection. According to clause 11,
The mixture of the white paper, turmeric and pine resin extracts is an animal feed additive for preventing or improving symptoms of avian influenza virus infection, characterized in that the white paper, turmeric and resin extracts are in a weight ratio of 1 to 5:1 to 5:0.1 to 0.5. According to any one of claims 11 to 13,
An animal feed additive for preventing or improving symptoms of avian influenza virus infection, characterized in that the mixture of the white paper, turmeric and pine resin extracts further includes vitamin C and chitosan.