KR20120132757A - Lipophilic fractions of green tea and green tea by product, and compound from the same with anti-influenza activity - Google Patents

Abstract

PURPOSE: An agent containing glycerol glycolipid compounds for preventing and treating influenza diseases is provided to ensure excellent antiviral effect in vivo and in vitro and to be used in a veterinary composition, feed additives, and anti-viral disinfection composition for preventing and treating animal diseases caused by viral infection. CONSTITUTION: A glycerol glycolipid compound is denoted by chemical formula 1 or 2. A pharmaceutical composition for preventing and treating influenza diseases contains the glycerol glycolipid compounds and a pharmaceutically acceptable carrier. The influenza diseases are caused by influenza virus A. A composition for preventing and treating influenza virus diseases contains a fat-soluble extract of green tea as an active ingredient. The fat soluble extract is prepared by hexane or dichloromethane. A veterinary composition for preventing and treating the influenza diseases contains the glycerol glycolipid compounds. A feed additive composition contains the glycerol glycolipid compounds.

Description

Lipid fractions of green tea and green tea by-products having anti-influenza activity and glycerol glycolipid compounds isolated therefrom {Lipophilic fractions of green tea and green tea by product, and compound from the same with anti-influenza activity}

The present invention relates to a fat-soluble fraction of green tea and green tea by-products having anti-influenza activity and a glycerol glycolipid compound isolated therefrom.

Influenza virus is an RNA virus belonging to the family Orthomyxoviridae, and its serotypes are classified into three types, type A, type B and type C. Among them, hepatitis B and C have been identified only in humans, while hepatitis A has been identified in humans, horses, pigs, other mammals and various types of poultry and wild birds (Selmons et al., Avian Dis. , 18 (1), pp119-124, 1974; Turek R et al., Acta Virol., 27 (6), pp523-527, 1983; Webster RG et al., Microbiol Rev., 56 (1), pp152- 179, 1992).

The serotypes of influenza A viruses are classified according to the two types of proteins on the surface of the virus: hemagglutinin (HA) and neuraminidase (NA), and 144 types (HA proteins) 16 species and 9 NA proteins). HA acts as a virus to attach to somatic cells, and NA allows the virus to penetrate into cells. (Alexander DJ, Vet. Microbiol., 74 (1-2), pp3-13, 2000).

The normal natural host of influenza A virus is known as wild waterfowl such as ducks and seagulls.As a result of epidemiological investigations of influenza infection in wild birds around the world, there are 16 existing HA and 9 NA influenza species. It has been confirmed that viruses are infected in wild birds (Selmons et al., Avian Dis., 18 (1), pp119-124, 1974). Avian influenza virus, classified as type A, is a common infectious disease virus, a non-pathogenic avian influenza that causes mild respiratory symptoms when infected with chickens. Low pathogenic avian influenza (LPAI) and high pathogenic avian influenza (HPAI) with over 95% high mortality and also called "bird flu". (Alexander DJ, Vet. Microbiol., 74 (1-2), pp3-13, 2000). The highly pathogenic avian influenza is classified as Class A by the International Water Office (OIE) and domestically infected with animal species I.

Humans are hosts of influenza A, B and C viruses. History has recorded three influenza pandemics by serotypes H1N1, H2N2 and H3N2. The Spanish flu, the first pandemic of influenza, has been documented as one of the greatest damages to humanity, and between 20 and 50 million people died during that period (Walter JH, Bull. NY Acad. Med., 54, pp 855-864). , 1978). The causative virus of the Spanish flu turned out to be serotype A / H1N1, which is very similar to the swine influenza virus. In the third influenza pandemic, the causative virus originated from birds and caused damage. In 2009, the new influenza virus originated from pigs, resulting in more than 130,000 infections and more than 800 deaths in more than 100 countries. If the resulting influenza virus is infectious to humans, it is highly likely to cause such damage.

In order to prevent such damages, the development of vaccines and therapeutics has been actively conducted worldwide, and has been commercialized and applied to humans and animals. The vaccine has been developed and used as a deadly vaccine and a recombinant vaccine, but due to the characteristics of the influenza virus that is easily mutated, the strain isolated in the past has a disadvantage in that the protection against the recent strain is reduced. As a therapeutic agent, amantadine, rimantadine, zanamivir and oseltamivir are used, but there are side effects such as the emergence of resistant strains, vomiting and dizziness. .

Green tea is used by processing the leaves of the evergreen shrub (Thea sinensis L. = Camellia sinensis), which belongs to Theaceae, or the leaves of its cultivated varieties.It is used for manufacturing raw materials of caffeine, palates, various foods, etc. Use as a diuretic, exciting, astringent medicine.

The leaves of the tea tree are lanceolate, elliptical, or long oval, and the edges of the leaves are slightly sharp, the petiole is short, and the leaf is 10cm long and 5cm wide with dull teeth. Young leaves have nail-like protrusions on the tip, single cells on the epidermis on the epidermis, and the side veins are slightly obtuse with the main vein, and the taste is astringent and bitter. In Korea, tea trees are grown mainly in Jeju Island and southern regions.

Green tea contains phenolic compounds (+)-catechin, (-)-epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin gallate, (-)-epigallocatechin, theaflavin, and caffeine, purine alkaloid. xanthine and theophylline have been reported.

As physiological activity of green tea, anti-inflammatory, antioxidant, anti-cancer activity, nerve excitement, cardiac, diuretic effect, and catechins, the main component, have been reported anti-cancer activity and angiogenesis inhibitory activity.

Green tea by-products are the residues left after hot water extraction of green tea, which is low in economic value and suitable for use as a raw material. Extracting green tea by-products with lower (C1 ~ C4) alcohols and fractionating organic solvents have a relatively high content of fat-soluble fractions with less catechin components. A large number of studies on catechin, the main component, have been reported, but few studies have been conducted on glycerol glycolipids isolated from fat-soluble fractions.

The present invention solves the above problems and the object of the present invention is to provide an anti-influenza formulation comprising a novel plant extract.

Another object of the present invention is to provide an anti-influenza preparation containing a compound derived from a plant extract as an active ingredient.

It is another object of the present invention to provide a method for separating a compound capable of treating or preventing influenza diseases from the fat-soluble fraction of green tea and / or green tea by-products.

In order to achieve the above object, the present invention provides a glycerol glycolipid compound selected from the group consisting of the following general formula (1) and (2).

[Formula 1]

[Formula 2]

In another aspect, the present invention provides a pharmaceutical composition for the prevention and treatment of influenza diseases comprising the compound of the present invention and a pharmaceutically acceptable carrier.

In one embodiment of the present invention, the influenza disease is preferably caused by influenza virus A, but is not limited thereto.

In another aspect, the present invention provides a composition for the prevention and treatment of influenza diseases comprising the fat-soluble extract of green tea as an active ingredient.

In one embodiment of the present invention, the fat-soluble extract is preferably extracted with a hexane or dichloromethane solvent, but is not limited thereto. Green tea or green tea by-products without direct extraction of lower alcohols (C1 to C4) hexane or dichloromethane It is possible to obtain a fat-soluble extract by extraction with the solvent used is not limited to hexane and dichloromethane.

In addition, the present invention a) extract the plant obtained after hot water extraction of green tea or green tea with methanol, concentrated and partitioned into hexane to obtain a hexane fraction, b) the hexane fraction on the column hexane / ethyl acetate, dichloromethane / methanol, It provides a method of obtaining the compound of the present invention comprising the step of eluting methanol sequentially with a solvent.

In one embodiment of the invention, the method preferably further comprises the step of performing column chromatography with a dichloromethane / methanol / water solvent after step b), but is not limited thereto.

In one embodiment of the present invention, the fat-soluble extract is preferably extracted with a hexane or dichloromethane solvent, but is not limited thereto. Green tea or green tea by-products without direct extraction of lower alcohols (C1 to C4) hexane or dichloromethane It is possible to obtain a fat-soluble extract by extraction with the solvent used is not limited to hexane and dichloromethane.

In addition, the present invention a) extract the plant obtained after hot water extraction of green tea or green tea with methanol, concentrated and partitioned into hexane to obtain a hexane fraction, b) the hexane fraction on the column hexane / ethyl acetate, dichloromethane / methanol, It provides a method of obtaining the compound of the present invention comprising the step of eluting methanol sequentially with a solvent.

In one embodiment of the invention, the method preferably further comprises the step of performing column chromatography with a dichloromethane / methanol / water solvent after step b), but is not limited thereto.

The present invention provides a veterinary composition for the prevention and treatment of diseases caused by viruses of animals comprising the fat-soluble fraction of green tea and green tea by-products having anti-influenza activity and the glycerol glycolipid compound isolated therefrom as an active ingredient.

Diseases caused by in vivo infection of the influenza virus family viruses as defined herein include avian influenza or variant infectious avian influenza.

The present invention also provides an animal feed additive for preventing and improving diseases caused by a virus containing a fat-soluble fraction of green tea and green tea by-products having anti-influenza activity and a glycerol glycolipid compound isolated therefrom as an active ingredient, and a feed comprising the same. do.

In addition, the present invention is a veterinary composition for the prevention and treatment of diseases caused by the anti-influenza virus of animals other than humans containing fat-soluble fractions of green tea and green tea by-products and glycerol glycolipid compounds isolated therefrom are added to the total weight of the composition. 0.1 to 50% by weight of the extract.

Hereinafter, the present invention will be described in detail.

Compounds derived from green tea and green tea by-products of the present invention can be used safely in long-term administration for the purpose of prevention because the active ingredient has little toxicity and side effects. The veterinary composition comprising the compound derived from the green tea of the present invention as an active ingredient may further comprise suitable excipients and diluents according to conventional methods.

Excipients and diluents that may be included in the veterinary composition comprising the green tea and green tea by-products of the present invention as an active ingredient include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch , Acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, three Ethanol, stearyl alcohol, liquid paraffin, sorbitan monostearate, polysorbate 60, methyl paraben, propyl paraben and mineral oil.

The veterinary composition comprising a compound derived from green tea and green tea by-products according to the present invention as an active ingredient may further include fillers, anti-coagulants, lubricants, wetting agents, spices, emulsifiers, preservatives, etc. Medical compositions may be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after it is administered to an animal, and the formulation may be a powder, granule, tablet, capsule, suspension emulsion , Solutions, syrups, aerosols, soft or hard gelatin capsules, suppositories, sterile injectable solutions, sterile external preparations, and the like.

The veterinary composition according to the present invention may vary depending on the age, sex and weight of the animal, but may be administered once to several times in an amount of 0.1 to 100 mg / kg. In addition, the dosage of the compound derived from the green tea and green tea by-products may be increased or decreased depending on the route of administration, the degree of disease, sex, weight, age, and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The present invention also provides an animal feed additive and a feed comprising the same, which are useful for the prevention and amelioration of diseases caused by viruses comprising the compounds derived from green tea and green tea by-products as active ingredients.

The green tea extract for animal feed additives may be 20 to 90% high concentrate or powder or granule form.

Compounds derived from green tea and green tea by-products for animal feed additives of the present invention include organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate and polyphosphate (polymeric phosphate). Or any one or more of natural antioxidants such as polyphenols, catechin, alpha-tocopherol, rosemary extract, vitamin C, pine needle extract, licorice extract, chitosan, tannic acid, phytic acid, and the like. can do.

Animal feed additives containing the compounds derived from green tea and green tea by-products of the present invention and feed containing them include amino acids, inorganic salts, vitamins, antibiotics, antimicrobials, antioxidants, antifungal enzymes, live microbial preparations, and the like as auxiliary ingredients. Cereals such as wheat, oats, barley, corn and rice, in which various auxiliary agents are crushed or crushed; Vegetable protein feed, such as rape, soybeans and sunflower; Animal protein feeds such as blood meal, meat meal, bone meal and fish meal; Sugars and dairy products, for example, a dry ingredient consisting of various powdered milk and whey powder, and a drying additive, all mixed together with a liquid component, and a component which becomes a liquid after heating, that is, a lipid, for example, an animal liquefied by heating. In addition to the main components such as fats and vegetable fats can be used with substances such as nutritional supplements, digestion and absorption enhancers, growth promoters, disease prevention agents and the like.

The compound derived from the green tea and green tea by-products for the animal feed additives may be administered to animals alone and in combination with other feed additives in an edible carrier.

In addition, the compounds derived from the green tea and green tea by-products for animal feed additives can be easily administered as a top dressing or directly mixed into animal feed or separately from the feed, in a separate oral formulation, by injection or transdermal or in combination with other ingredients. can do. Typically, single or divided daily dosages can be used as is well known in the art.

When the compound derived from the green tea for animal feed additives is administered separately from the animal feed, the dosage form of the extract, as is well known in the art, is an immediate release or sustained release formulation in combination with a non-toxic pharmaceutically acceptable edible carrier. It can be prepared by. Such edible carriers may be solid or liquid, for example corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the dosage form of the extract may be tablets, capsules, powders, torokies or sugar-containing tablets or top dressings in microdisperse form. If a liquid carrier is used, it may be in the form of soft gelatin capsules or syrups or liquid suspensions, emulsions or solutions. In addition, the dosage form may contain auxiliaries such as preservatives, stabilizers, wetting or emulsifying agents, solution promoters and the like. They may also contain other substances useful for the improvement and prevention of diseases caused by viruses.

In addition, animal feed comprising compounds derived from green tea and green tea by-products as animal feed additives may be any protein-containing organic cereal meal commonly used to meet the dietary needs of animals. Such protein-containing flours typically consist mainly of corn, soy flour, or corn / bean flour mixtures.

The animal feed additive may be used by adding to the animal feed by dipping, spraying or mixing.

The invention is applicable to a number of animal diets, including mammals, poultry and fish. More specifically, the diet is commercially important mammals such as pigs, cows, sheep, goats, laboratory rodents (rats, mice, hamsters and gerbils), furry animals (eg, mink and fox), and Zoo animals (eg monkeys and tailless monkeys), as well as livestock (eg cats and dogs). Commercially important poultry typically include chickens, turkeys, ducks, geese, pheasants and quails. Commercially raised fish, such as trout, may also be included.

In addition, feed mixtures containing compounds derived from green tea and green tea by-products are thoroughly mixed and, depending on the degree of pulverization of the components, a viscous granulated or granular material is obtained. It is fed as a mash or formed into the desired discrete shape for further processing and packaging. At this time, in order to prevent separation during storage, it is preferable to add water to the animal feed, followed by a conventional pelleting, expansion, or extrusion process. Excess water may be removed to dryness.

The present invention also provides an anti-influenza virus disinfecting composition containing a compound derived from green tea and green tea by-products as an active ingredient.

The fat-soluble fraction derived from the green tea of the present invention and the compound containing the same can be used for in vitro experiments using influenza A viruses and in vitro experiments using the virus-infected animals. Since the antiviral effect by the killer virus is excellent, it can be used in veterinary compositions, feed additives and killer virus disinfectant compositions for the prevention and treatment of animal diseases caused by the infection of the virus in the body.

Figure 1 is a table showing the in vitro efficacy test results of green tea and green tea by-product extract using neutral red colorimetric analysis.
Figure 2 is a table showing the effect of inhibiting virus growth in lungs when the green tea and green tea by-products administration to BALB / c mice.
Figure 3 is a table showing the virus re-separation results when administration of green tea by-products to SPF chicken.
Figure 4 is a schematic diagram of the extraction and organic solvent fractionation process of the green tea by-product,
Figure 5 is a schematic diagram of the separation process of the compound from the hexane fraction of the green tea by-product,
6 shows the molecular structure of GT-lipid glycoside II,
7 shows the molecular structure of GT-lipid glycoside V,
8 shows the inhibitory effect of the green tea by-product fat-soluble fraction on Influenza A / NWS / 33 (H1N1) virus,
Figure 9 shows the inhibitory effect of Influenza A / NWS / 33 (H1N1) virus of the compound isolated from the green tea by-product fat-soluble fraction.

Hereinafter, the present invention will be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the invention and the scope of the invention is not to be construed as being limited by the following examples.

Example  1: of green tea and green tea by-product extract In vitro Anti-influenza efficacy  exam

The anti-influenza efficacy of catechin, the main ingredient of green tea extract, is already known. In order to demonstrate the anti-influenza efficacy of green tea by-products by comparing the in vitro anti-influenza efficacy of green tea extract and green tea by-product extract, neutral red colorimetric analysis was performed. As a result of the test of green tea extract, green tea extract and green tea by-product extract showed similar selection index, indirectly confirming that the active ingredients in green tea remained in the green tea by-products (FIG. 1).

In general, 50% reduction in viral release is 50% effective concentration (EC50) and 50% reduction in cell survival is 50% cytotoxic concentration (C50). The SI (selectivity index) is CC50 / EC50. The larger the SI value, the greater the virus growth inhibition effect.

Example  2: Green Tea Byproduct Using Mouse Model Anti-influenza  Efficacy test

In vitro anti-influenza efficacy of green tea extract and green tea by-product extract was compared to test the influenza efficacy of green tea byproducts. As a result of administering the green tea by-product extract to the virus-infected BALB / c mice, it was confirmed that the virus content in the lungs was significantly decreased after 3 days of challenge compared to the negative control group not receiving the extract (FIG. 2).

Example  3: SPF  Green Tea By-product Using Chicken Model Anti-influenza  Efficacy test

In order to demonstrate the anti-influenza efficacy of green tea by-products by comparing the in vitro anti-influenza efficacy of green tea extract and green tea by-product extract, an animal test using SPF chicken was conducted. Green tea by-product extracts were administered as feed additives or negative additives to reduce the virus resegregation rate in tracheal and caecum tonsil at all concentrations and significantly reduced the titer of re-separated virus to confirm the anti-influenza efficacy of green tea by-products. In particular, considering that the high titer virus released by feces from influenza infected chickens plays an important role in the spread of influenza, it is judged that the green tea by-products have a high industrial use value (FIG. 3).

Example  4: Extraction of Green Tea By-Products and Organic Solvent Fraction

2 kg of green tea was extracted twice with 20 L of distilled water in a 90 ° C. water bath, and concentrated to obtain a water extract.

The hydrothermally extracted plant was dried, extracted with 7.5 L of methanol three times at 55 ° C., and concentrated to obtain a methanol extract of green tea by-product. The methanol extract 143g was suspended in 90% methanol, and then partitioned three times with the same amount of hexane to obtain a hexane layer. The remaining 90% methanol layer was concentrated and again suspended in 1.4 L of 70% methanol and partitioned three times with dichloromethane to obtain a dichloromethane fraction. The 70% methanol layer was concentrated and suspended again in 1.4 L of distilled water and partitioned three times sequentially with ethyl acetate and butanol to obtain an ethyl acetate, butanol and water fraction.

Example  5: green tea by-product Hexane  Separation of Compounds from Fractions

We tried to isolate the active ingredient from the hexane fraction, one of the green tea by-product fat-soluble fractions, which showed significant antiviral effects (EC50: 29.77μg / ml; SI: 18.50) against Influenza virus A / NWS / 33 (H1N1). The hexane fraction of the green tea by-product was sequentially elution of hexane / EtOAc = 10: 1, 3: 1, 1: 1, CH2Cl2 / MeOH = 2: 1, 1: 1, MeOH with solvent on a silica gel column (1-11) was obtained. Small fraction 11 was divided into 10 fractions (11-A through 11-J) by silica gel column chromatography, and fractions 11-F and 11-G were separated by CH2Cl2 / MeOH / H2O = 60: 10: 1 and 40:10. Two kinds of galactosylacylglycerol, lipid glycosides, were obtained by silica gel column chromatography using 1: 1 solvent. (Fig. 5)

Example  6: Hexane  Structure of Compounds Isolated from Fractions

Of the two lipid glycosides isolated from the active subfraction, GT-lipid glycoside Ⅱ is digalactosylmonoacylglycerol, and galactose 2 mole is bound to glycerol at position 1 and fatty acid is lauric acid to glycerol at position 3. I could see that. GT-lipid glycoside V is a monogalactosylmonoacyl glycerol compound with unsaturated fatty acids. The unsaturated fatty acid of V was identified as arachidonic acid with four double bonds.

Thus, compounds GT-lipid glycosides II and V are glycerol 3-O-β-galactopyranosyl- (1 → 6) -β-galactopyranosyl 1-O-lauric acid ester and glycerol 3-O-β-galactopyranosyl 1-O-arachidonic, respectively. Determined by acid ester.

1.GT-lipid glycoside II (FIG. 6)

ESI-MS (positive) m / z: 605 [M + Na] +

ESI-MS (negative) m / z: 581 [M-H]-

1 H-NMR (CDCl 3, 500 MHz) δ:

0.89 (3H, t, J = 7.5 Hz), 1.37 (brs), 1.57 (brs), 2.34 (brs), 3.40-5.35 (Gal-H and glycerol-H)

13 C-NMR (CD3OD, 125 MHz) δ:

(fatty acid)

14.3, 22.9, 25.1, 29.5-30.0, 32.1, 34.4, 174.0

(galactose and glycerol)

62.0, 63.1, 68.3, 68.5, 69.1, 70.2, 70.3, 70.5, 70.8, 71.2, 71.4, 72.8, 73.4, 99.9, 104.1

2.GT-lipid glycoside V (FIG. 7)

ESI-MS (positive) m / z: 563 [M + Na] +

ESI-MS (negative) m / z: 539 [M-H]-

1H-NMR (CD3OD, 500MHz) δ:

0.88 (3H, t, J = 7.0 Hz), 1.25 (brs), 1.57 (brs), 2.0-2.15 (m),

2.29 (brs), 2.79 (brs), 3.20? 4.50 (Gal-H and glycerol-H), 5.35 (8H, m)

13 C-NMR (CD3OD, 125 MHz) δ:

(fatty acid)

14.3, 22.9, 25.2, 29.5-30.1, 32.2, 34.4, 127.3, 128.0, 128.4, 128.5, 129.8, 130.2, 130.4, 132.1, 174.2

(Galactose and glycerol)

63.2, 63.4, 66.6, 68.4, 70.6, 71.3, 71.9, 73.8, 99.2

Example  7: green tea by-product Fat-soluble component Anti-influenza  activation

The soluble and dichloromethane fractions obtained from methanol extracts of green tea by-products were EC ₅₀ of 59.59 (SI = 11.13) and 8.34 (SI = 5.21) ug / ml for influenza A / NWS / 33 (H1N1) virus, respectively. The value is shown. (Figure 8)

Example  8: of isolated compound Anti-influenza  activation

The hexane fraction of the green tea by-product was subjected to active-oriented separation to separate the compounds. The results of measuring the activity of influenza A / NWS / 33 (H1N1) virus using the CPE inhibitory assay as the isolated compounds are shown in the following table. The EC ₅₀ values were 99.02 and 91.30 μg / ml in GT-lipid glycoside Ⅱ and Ⅴ, respectively, and the SI values were 1.68 and 3.36, respectively. (Fig. 9)

In general, 50% reduction in viral release is 50% effective concentration (EC50) and 50% reduction in cell survival is 50% cytotoxic concentration (C50). The SI (selectivity index) is CC50 / EC50. The larger the SI value, the greater the virus growth inhibition effect.

Claims (10)

A glycerol glycolipid compound selected from the group consisting of Formula 1 and Formula 2.

[Formula 1]

(2) A pharmaceutical composition for preventing and treating influenza diseases comprising the compound of claim 1 and a pharmaceutically acceptable carrier. The composition for preventing and treating influenza diseases according to claim 2, wherein the influenza disease is caused by influenza virus A. A composition for the prevention and treatment of influenza diseases comprising fat-soluble extract of green tea as an active ingredient. The method of claim 4, wherein the fat-soluble extract is a composition for the prevention and treatment of influenza diseases, characterized in that extracted with hexane or dichloromethane solvent. a) the plant obtained after hot water extraction of green tea or green tea is extracted with an alcohol selected from the group consisting of methanol, ethanol, propanol and butanol, concentrated and partitioned into hexane to obtain a hexane fraction,
b) obtaining the compound of claim 1, comprising eluting the hexane fraction sequentially on a column with hexane / ethyl acetate, dichloromethane / methanol and methanol as a solvent. The method of claim 6, wherein the method further comprises performing column chromatography with a dichloromethane / methanol / water solvent after step b). Veterinary composition for the prevention and treatment of influenza diseases comprising the compound of claim 1. Feed additive composition comprising the compound of claim 1. A composition for disinfecting virin comprising a compound of claim 1.

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