KR101334143B1 - Composition comprising extract of polygala karensium or xanthone compounds isolated therefrom for treating or preventing cold, avian influenza, swine influenza or novel influenza - Google Patents

Abstract

The present invention relates to a composition containing a Polygala karensium extract and xanthone-based compounds isolated from the same for preventing or treating flu, avian influenza, swine influenza, or novel swine-origin influenza. The Polygala karensium extract and xanthone-based compounds isolated from the same effectively and non-selectively suppress neuraminidase of flu virus, avian influenza virus, swine influenza virus, novel swine-origin influenza, and Tamiflu resistant novel swine-origin influenza virus, thereby being used as a therapeutic agent for treating flu, avian influenza, swine influenza, and swine-origin influenza and being applied in cosmetic products, health foods, animal feeds, and an animal medicinal industry. [Reference numerals] (AA) Swine influenza virus (H1N1);(BB,HH,NN,TT) Compound 1;(CC,II,OO,UU) Compound 2;(DD,JJ,PP,VV) Compound 3;(EE,KK,QQ,WW) Compound 4;(FF,LL,RR,XX) Compound 5;(GG) Avian influenza virus (H9N2);(MM) Novel swine-origin influenza virus (novel H1N1);(SS) Novel swine-origin influenza virus resistant to Tamiflu (H1N1[H274Y])

Description

Composition comprising extract of Polygala karensium or xanthone compounds isolated therefrom for treating or preventing Cold containing polygala carenium extract and xanthone-based compounds isolated therefrom , Avian influenza, Swine influenza or Novel influenza}

The present invention relates to a composition for the prevention or treatment of cold, avian influenza, swine influenza or swine flu containing Polygala karensium extract and xanthones-based compounds isolated therefrom.

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

There are two proteins on the surface of the influenza virus: hemagglutinin (HA) and neuraminidase (NA) .There are 16 types of HA and 9 types of NA, so 16 × 9 = 144 types of influenza viruses May occur. Influenza infections in birds are mainly related to H5, H7 and H9 types, with only three types of hemagglutinin (H1, H2, H3) and two types of neuraminidase (N1 and N2) in humans. In principle, humans are not infected with avian influenza, as it causes infection. Recently, however, avian influenza has occurred in which avian influenza virus is directly transmitted to humans without undergoing genetic recombination.

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

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

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

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

On the other hand, the common cold caused by influenza virus is the common influenza virus (type A, B, C, adenovirus, corona virus (corona virus), rhinovirus (rhinovirus) , RS virus (respiratory syncytial virus) due to a variety of causes, such as disease is difficult to treat, prevent, vaccine manufacturing. Therefore, there is an urgent need to develop a therapeutic agent that can effectively treat various viruses that can cause colds.

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

In the present invention, a cold, bird influenza, swine flu and swine flu against swine flu, swine flu, swine flu, and swine flu Neuraminidase inhibitors were searched. In general, finding new active ingredients from natural medicines used in traditional medicine has many advantages over experimental modification of existing drugs to develop new ingredients of medicine. In particular, since these active ingredients have been used for a long time, there is little concern about toxicity due to drugs. This fact can be confirmed from the development of Tamifludo starting material, which is used as a treatment for swine flu, as the main ingredient of 'Staranis' fruit, which is an indigenous plant in China.

There are about 500 species of plants in the Polygala genus, and they are effective in amnesia, colds, bronchitis, and nervous breakdown. Especially, the triterpenoid saponins and xanthone compounds in the plants of Polygala genus have various biological activities. (Phytochemistry Reviews, 2005, 4, 139-149; Phytochemistry 2008, 69, 1617-1624; Chem. Pharm. Bull., 2002, 50, 1499-1501; Nat. Prod. 2006, 69, 1305 1309; Planta Med., 2008, 74, 133-141).

In the present invention, by separating the Xanthone-based compounds from polygala carenium, the polygala carenium extract and these compounds inhibit the neuraminidase of avian influenza virus, swine influenza virus, swine flu virus and Tamiflu-resistant swine flu virus It was confirmed.

Lacaille-Dubois, M. A. et al, Triterpene saponins from Polygalaceae., Phytochemistry Reviews, 2005, 4, 139-149. Fu, J. et al, Oligosaccharide polyester and triterpenoid saponins from the roots of Polygala japonica., Phytochemistry 2008, 69, 1617-1624. Acqua, S. D. et al, Cytotoxic compounds from Polygala vulgaris., Chem. Pharm. Bull., 2002, 50, 1499-1501. Cheng, M. C. et al., Antidepressant principles of the roots of Polygala tenuifolia., Nat. Prod. 2006, 69, 1305-1309. Li, C. et al., Triterpenoid saponins with neuroprotective effects from the roots of Polygala tenuifolia., Planta Med., 2008, 74, 133-141.

The object of the present invention is Polygala To provide a composition for the prevention or treatment of cold, avian influenza, swine influenza or swine flu containing karensium ) extract and xanthones-based compounds isolated therefrom.

1,3-dihydroxyxanthone (Compound 1 ), 1,7-dihydroxyxanthone (1,7-dihydroxyxanthone, Compound 2 ), 1,7-di Hydroxy-4-methoxyxanthone (1,7-dihydroxy-4-methoxyxanthone, compound 3 ), 1,3,7-trihydroxyxanthone (1,3,7-trihydroxyxanthone, compound 4 ), and, 1, 2,3,5-tetrahydroxyxanthone (1,2,3,5-tetrahydroxyxanthone, compound 5 ) containing at least one xanthone-based compound selected from the group consisting of Polygala ( Polygala) karensium ) relates to a composition for the prevention or treatment of colds, avian influenza, swine influenza or swine flu.

[Formula 1]

The polygala calenium extract may be prepared by extracting with water, lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, chloroform or a mixed solvent thereof.

In addition, the present invention is 1,3-dihydroxyxanthone (Compound 1 ), 1,7-dihydroxyxanthone (Compound 2 ), 1,7-dihydroxy-4-methoxyxanthone (Compound 3 ) containing 1,3,7-trihydroxyxanthone (compound 4 ), and one or more xanthene compounds selected from the group consisting of 1,2,3,5-tetrahydroxyxanthone (compound 5 ) It relates to a composition for the prevention or treatment of colds, avian influenza, swine influenza or swine flu.

The present invention also provides 1,3-dihydroxyxanthone (Compound 1 ), 1,7-dihydroxyxanthone (Compound 2 ), 1,7-dihydroxy-4-methoxyxanthone (Compound 1 ) 3 ), 1,3,7-trihydroxyxanthone (compound 4 ), and 1,2,3,5-tetrahydroxyxanthone (compound 5 ) include one or more xanthone compounds selected from the group consisting of Done It provides a dietary supplement for the prevention or amelioration of a cold, avian influenza, swine influenza or swine flu containing polygala calenium extract.

On the other hand, the present invention provides an animal drug or feed additive containing the polygala carenium extract and the xanthone-based compound separated therefrom. In addition, the present invention may provide a natural antiseptic agent containing the polygala carenium extract and a xanthone compound separated therefrom.

Hereinafter, the present invention will be described in detail.

Polygalar carenium extract of the present invention, 2 to 50 times (w / v) water of polygalar carnsium, lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, chloroform or a mixed solvent thereof Can be prepared by extraction. The lower alcohol may be selected from ethanol, methanol, propanol, isopropanol and butanol.

In addition, the extract of the polygala carenium extract of the present invention by an organic solvent (alcohol, aqueous alcohol solution, ethyl acetate, acetone, chloroform, etc.), hexane and water distribution, method of using adsorption resin, such as diion HP-20 resin, The active compound of the present invention can be easily obtained by combining alone or suitably a known method used for separation extraction of plant components, such as by column chromatography.

Preferably, 2-50 times (w / v) of n-hexane, ethyl acetate, n-butanol, and the like are sequentially added to the polygalar carenium extract to purify and fractionate it, followed by chromatography. It can be used to isolate the compounds having antiviral activity.

Chromatography used in the present invention includes silica gel column chromatography, LH-20 column chromatography, thin layer chromatography (TLC) and high performance liquid chromatography high performance liquid chromatography, etc.) can be used.

Determination of the activity of the polygala carenium extract of the present invention and the xanthone-based compound isolated therefrom is performed by using the polygala carenium as water, lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, chloroform or a mixed solvent thereof. Extracting and fractionating with ethyl acetate; Separating and purifying the xanthone compound using pure chromatography; Identifying a xanthone compound through the chemical structure of the separated active compound; And measuring the inhibitory effect of the xanthone compound against avian influenza virus (H9N2), swine influenza virus (H1N1), swine flu virus (H1N1) neuraminidase and tamilflu resistant swine flu virus (H1N1). Step; can be confirmed through.

The pharmaceutical composition comprising the polygalal carenium extract according to the present invention and the xanthone-based compound isolated therefrom is orally prepared according to a conventional method such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, and the like. It may be used in the form of a dosage form, an external preparation, a suppository, and a sterile injectable solution. Carriers, excipients and diluents that may be included in the composition comprising the polygala carenium extract and the xanthone-based compound isolated therefrom 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 and Mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, for example, in the polygala carenium extract and the xanthone-based compound separated therefrom. Starch, calcium carbonate, sucrose or lactose, gelatin and the like are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

The dosage of the polygalar carenium extract and the xanthone-based compound isolated therefrom is determined by the age, sex and weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, the route of administration and the judgment of the prescriber. Will depend on. Dosage determinations based on these factors are within the level of ordinary skill in the art and generally the dosage ranges from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferable dosage is 0.1 mg / kg / day to 500 mg / kg / day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way. Polygala carenium extract of the present invention and the xanthone-based compound isolated therefrom can be administered to mammals such as rats, livestock, humans and the like by various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. Polygala carenium extract of the present invention and the xanthone-based compound isolated therefrom is almost no toxicity and side effects, so it is a drug that can be used safely even for long-term administration for the purpose of prevention.

In addition, the present invention provides a dietary supplement for the prevention of cold, avian influenza, swine influenza and swine flu, including the polygala calenium extract and food supplements. The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids. Examples of the foods to which the extract of the present invention can be added include various foods, beverages, gums, tea, vitamins , And health functional foods.

On the other hand, the present invention provides an animal drug or feed additive containing the polygala carenium extract and the xanthone-based compound separated therefrom. The animal drugs and feed additives can also be used for infectious enteritis caused by bacterial infection, which is a common common disease related to neuraminidase, which commonly occurs in animals and humans.

In addition, the present invention may provide a natural antiseptic agent containing the polygala carenium extract and a xanthone compound separated therefrom.

The present invention relates to a composition for the prevention or treatment of cold, avian influenza, swine influenza or swine flu, containing Polygala karensium extract and xanthone-based compounds isolated therefrom. The composition of the polygala carenium extract and the xanthone-based compound isolated therefrom is excellent in the effect of non-selectively inhibiting the neuraminididae of avian influenza, swine influenza, virus, swine flu virus, Tamiflu-resistant swine flu virus Can be used as a treatment for avian influenza, swine influenza, swine flu-related diseases, and can be applied to cosmetics, health food, animal feed, animal medicine industry, and the like.

FIG. 1 shows the HPLC spectral results obtained by analyzing Compounds 1 to 5 isolated from Polygala Carenium.
Figure 2 shows the results confirming the neuraminidase inhibitory activity of the influenza of avian influenza virus, swine influenza virus, swine flu, Tamiflu-resistant swine flu virus against the compounds 1 to 5 of the present invention.
FIG. 3A is a Lineweaver Burk plots plot showing that Compound 3 of the present invention inhibits neuraminidase of H1N1 as a noncompetitive inhibitor, and FIG. 3B is a Dixon plot plot.
4 shows the results of the cell lesion effect recovery assay, FIG. 4A shows that the virus survival of MDCK cells treated with 5 to 5 μg / ml of the compound 1 to 5 of the present invention is recovered by virus. FIG. 4B shows that the compound 3 of the present invention is treated to MDCK cells by concentration. 4C shows that the cell morphology is similar to Tamiflu-treated cells when 5 mg / ml of the compound 3 of the present invention is treated to cells treated with porcine influenza virus.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art.

<Example 1: Confirmation of Solvent Extraction Conditions of Active Compounds from Polygalar Carencium>

Polygala Karensium was collected in Lao Cai province, Vietnam in 2010. After drying the roots of Polygala Karensium, the dried roots were used in the present invention. 10 g of the root dry matter of the polygalar carenium was refluxed three times for 4 hours using 50 ml of a solvent such as distilled water, ethanol, methanol, acetone, ethyl acetate, chloroform, and the like. Thereafter, the extract was removed under reduced pressure, and the extract was concentrated under reduced pressure to obtain 0.7-1.5 g of extract per extract (however, the water extract was extracted at 90 ° C. for 8 hours and then the filtrate was lyophilized). In addition, the activity of each extract (final concentration of 20 µg / ml) against neuraminidase was confirmed using the swine influenza virus culture solution of Example 4-2 and the method of Example 6, and the results are shown in Table 1 below. It was. Referring to the results of Table 1, the extract of Polygala carensium was good activity under all conditions, but the activity of the methanol or ethanol extract was the highest and set this as the optimum extraction conditions.

Condition (20 µg / ml) Of swine influenza virus (H1N1) compared to untreated group
Inhibitory Activity against Neuraminidase 70% ethanol extract 33.4% 70% methanol extract 35.2% Ethanol extract 40.4% Methanol extract 41.2% Acetone extract 30.1% Ethyl acetate extract 29.1% Chloroform extract 25.2% Distilled water extract 4.01%

< Example  2 : Poligala Karensium  From the extract Xanthone  Separation of compounds &gt;

4 kg of methanol was added to 2 kg of dried roots of polygalar carenium and extracted at room temperature. This process was performed a total of three times. The extract was concentrated under reduced pressure to prepare a dried product. The dried methanol extract (230.0 g) was suspended in 2 L of water, and each fraction was sequentially partitioned into 1.5 L of n-hexane, ethyl acetate, and n-butanol. Each fractionation step was repeated three times.

The neuraminidase activity of the avian influenza virus, the swine influenza virus, and the Tamiflu-resistant swine flu virus were measured for each solvent fraction, and the ethyl acetate fraction and butanol fraction showed better activity. IC ₅₀ = 38.24 ± 2.78 μg / ml). Neuraminidase activity was confirmed by the method of Example 6 using the swine influenza virus culture of Example 4-2.

The ethyl acetate fraction (46.0 g) was chromatographed on a TLC-based silica gel column (10 × 25 cm; 63200 μm particle size) and eluted with a concentration gradient of n-hexane / EtOAc 9: 1 → 1: 9 [2.5 L]. 6 small fractions (F1 to F6) were obtained. Among them, additional fractions were performed for F2 (5.2 g), F4 (4.8 g) and F5 (6.7 g) which have good inhibitory activity against neuraminidase of swine influenza virus. F2 subfractions were divided into 6 subfractions (F2.1F2.6) using RP-18 column (5 × 30 cm; 4063 μm particle size) and MeOH / H ₂ O (3: 1 → 10: 1) gradient elution conditions. Among them, the F2.3 (120 mg) subfraction was purified by HPLC [Optima Pak C18 column (10 × 250 mm, 10 μm particle size, RS Tech, Korea); mobile phase MeOH in H ₂ O containing 0.1% HCO ₂ H (065 min: 64% MeOH, 6570 min: 64100% MeOH, 7080 min: 100% MeOH); flow rate 2 ml / min; Compound 1 (tR = 45.5 min, 5.5 mg) was isolated using UV detection at 205 and 254 nm].

In small fraction F2.4 (560 mg), Compound 2 (145.0 mg) was isolated using a Sephadex LH-20 column (4 × 25 cm) and elution conditions with methanol.

In small fraction F4, seven small fractions were obtained using RP-18 column (5 × 30 cm; 4063 μm particle size) and concentration gradient elution conditions of MeOH / H ₂ O (1: 1 → 10: 1) (F4.1F4 .7). F4.4 small fraction (160.0 mg) was purified by HPLC (055 min: 50% MeOH, 60 min: 100% MeOH) to separate compound 3 (tR = 51.0 min, 22.0 mg). Small fraction F5 was obtained from four fractions (F5.1F5.4) using a Sephadex LH-20 column (7 × 30 cm) and methanol elution conditions, of which HPLC (045 min) was performed on F5.2 small fraction (180.0 mg). : 45% MeOH, 50min: 100% MeOH) was used to separate compound 4 (tR = 43.0 min, 11.0 mg). Compound 5 (tR = 50.0 min, 8.5 mg) was isolated by HPLC (055 min: 66% MeOH, 60 min: 100% MeOH) again in small fraction F5.3 (160.0 mg). HPLC spectra of the compounds 1 to 5 are shown in FIG. 1.

* HPLC conditions of FIG. 1

YMCC18 column: 250 × 4.6mm ID, particle size 6μm, Japan

wavelengths: 205 nm (blue line), 254 nm (red line)

Elution: MeOH in H ₂ O (040min: 50% MeOH; 4045min: 50100% MeOH; 4555min: 100% MeOH) at flow rate of 1ml / min

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

Example  3-1. 1,3- Dihydroxyxanthone  (Compound 1)

1,3-dihydroxyxanthone, yellow needles, mp 259260 ℃

UV (MeOH) λ _max 238, 325 nm

EIMS m / z 228 [M] ⁺

¹ H-NMR (500 MHz, acetone- d ₆ ) δ _H 12.90 (s, OH-1), 8.20 (1H, d, J = 8.5 Hz, H-8), 7.84 (1H, t, J = 8.0 Hz, H-6), 7.53 (1H, d, J = 8.5 Hz, H-5), 7.46 (1H, t, J = 8.0 Hz, H-7), 6.44 (1H, s, H-4), 6.27 ( 1H, s, H-2)

¹³ C-NMR (125 MHz, acetone- d ₆ ) δ _C 181.4 (C-9), 166.6 (C-3), 164.8 (C-1), 158.9 (C-4a), 156.9 (C-10a), 136.4 (C-6), 126.4 (C-7), 125.2 (C-8), 120.5 (C-8a), 118.6 (C-5), 103.9 (C-9a), 99.1 (C-2), 94.9 ( C-4).

Example  3-2. 1,7- Dihydroxyxanthone  (Compound 2)

1,7-dihydroxyxanthone, yellow needles, mp 195-196 ℃

UV (MeOH) λ _max 232, 309 nm

EIMS m / z 228 [M] ⁺

¹ H-NMR (500 MHz, acetone- d ₆ ) δ _H 12.71 (s, OH-1), 7.68 (1H, t, J = 8.5 Hz, H-3), 7.58 (1H, d, J = 2.5 Hz, H-8), 7.50 (1H, d, J = 8.5 Hz, H-5), 7.40 (1H, dd, J = 8.5, 2.5 Hz, H-6), 6.97 (1H, d, J = 8.5 Hz, H-4), 6.74 (1H, d, J = 8.5 Hz, H-2)

¹³ C-NMR (125 MHz, acetone- d ₆ ) δ _C 182.9 (C-9), 162.7 (C-1), 157.3 (C-4a), 154.9 (C-7), 151.0 (C-10a), 126.1 (C-6), 121.8 (C-8), 120.2 (C-5), 110.5 (C-2), 109.1 (C-8a, C-9a), 107.8 (C-4)

Example  3-3. 1,7- Dihydroxy -4- Methoxyxanthone  (Compound 3)

1,7-dihydroxy-4-methoxyxanthone, yellow needles, mp 239-240 ° C

UV (MeOH) λ _max 236, 324 nm

EIMS m / z 258 [M] ⁺

¹ H-NMR (500 MHz, acetone- d ₆ ) δ _H 12.11 (s, OH-1), 7.58 (1H, s, H-8), 7.56 (1H, d, J = 8.5 Hz, H-5), 7.42 (2H, d, J = 8.5 Hz, H-3, H-6), 6.68 (1H, d, J = 8.5 Hz, H-2), 3.95 (3H, s, OCH ₃ -4)

¹³ C-NMR (125 MHz, acetone- d ₆ ) δ _C 182.9 (C-9), 155.4 (C-1), 155.2 (C-7), 151.0 (C-10a), 149.0 (C-4a), 141.2 (C-4), 126.3 (C-6), 121.9 (C-8a), 121.2 (C-3), 120.5 (C-5), 109.7 (C-9a), 109.1 (C-8), 108.8 ( C-2)

Example  3-4. 1,3,7- Trihydroxyxanthone  (Compound 4)

1,3,7-trihydroxyxanthone, yellow needles, mp 298-299 ℃

UV (MeOH) λ _max 233, 308 nm

EIMS m / z 244 [M] ⁺

¹ H-NMR (500 MHz, acetone- d ₆ ) δ _H 12.98 (s, OH-1), 7.56 (1H, d, J = 2.5 Hz, H-8), 7.42 (1H, d, J = 8.5 Hz, H-5), 7.36 (1H, dd, J = 8.5, 2.5Hz, H-6), 6.49 (1H, s, H-4), 6.25 (1H, s, H-2)

¹³ C-NMR (125 MHz, acetone- d ₆ ) δ _C 181.3 (C-9), 166.4 (C-3), 164.7 (C-1), 159.1 (C-4a), 154.9 (C-7), 150.8 (C-10a), 125.1 (C-6), 121.9 (C-8), 120.0 (C-5), 109.4 (C-8a), 103.6 (C-9a), 98.8 (C-2), 94.6 ( C-4)

Example  3-5. 1,2,3,5- Tetrahydroxyxanthone  (Compound 5)

1,2,3,5-tetrahydroxyxanthone, yellow needles, mp 264-265 ℃

UV (MeOH) λ _max 250, 325 nm

EIMS m / z 260 [M] ⁺

¹ H-NMR (500 MHz, acetone- d ₆ ) δ _H : 12.98 (s, OH-1), 7.56 (1H, m, H-7), 7.43 (1H, m, H-8), 7.40 (1H, m, H-6), 6.26 (1H, s, H-4)

¹³ C-NMR (125 MHz, acetone- d ₆ ) δ _C : 181.2 (C-9), 152.5 (C-3), 152.0 (C-4a), 150.2 (C-1), 149.2 (C-5), 148.4 (C-10a), 136.3 (C-2), 122.5 (C-7), 120.1 (C-8a), 119.1 (C-6), 118.3 (C-8), 102.9 (C-9a), 99.5 (C-4).

< Example  4: Preparation of influenza virus

4-1. Avian Influenza (H9N2) Virus

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

4-2. Swine Influenza (H1N1) Virus (WT H1N1)

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

< Example  5: Neuraminidays Cloning >

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

Swine Flu Neuraminidase Peptide Sequences Used

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

MNPNQKIITIGSVCMTIGMANLILQIGNIISIWISHSIQLGNQNQIETCNQSVITYENNTWVNQTYVNISNTNFAAGQSVVSVKLAGNSSLCPVSGWAIYSKDNSVRIGSKGDVFVIREPFISCSPLECRTFFLTQGALLNDKHSNGTIKDRSPYRTLMSCPIGEVPSPYNSRFESVAWSASACHDGINWLTIGISGPDNGAVAVLKYNGIITDTIKSWRNNILRTQESECACVNGSCFTVMTDGPSNGQASYKIFRIEKGKIVKSVEMNAPNYHYEECSCYPDSSEITCVCRDNWHGSNRPWVSFNQNLEYQIGYICSGIFGDNPRPNDKTGSCGPVSSNGANGVKGFSFKYGNGVWIGRTKSISSRNGFEMIWDPNGWTGTDNNFSIKQDIVGINEWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPKENTIWTSGSSISFCGVNSDTVGWSWPDGAELPFTIDK

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

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

MNPNQKIITIGSVCMTIGMANLILQIGNIISIWISHSIQLGNQNQIETCNQSVITYENNTWVNQTYVNISNTNFAAGQSVVSVKLAGNSSLCPVSGWAIYSKDNSVRIGSKGDVFVIREPFISCSPLECRTFFLTQGALLNDKHSNGTIKDRSPYRTLMSCPIGEVPSPYNSRFESVAWSASACHDGINWLTIGISGPDNGAVAVLKYNGIITDTIKSWRNNILRTQESECACVNGSCFTVMTDGPSNGQASYKIFRIEKGKIVKSVEMNAPNY Y YEECSCYPDSSEITCVCRDNWHGSNRPWVSFNQNLEYQIGYICSGIFGDNPRPNDKTGSCGPVSSNGANGVKGFSFKYGNGVWIGRTKSISSRNGFEMIWDPNGWTGTDNNFSIKQDIVGINEWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPKENTIWTSGSSISFCGVNSDTVGWSWPDGAELPFTIDK

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

< Example  6: Of neuraminidays  Active measurement>

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

Inhibition rate: [{C- (A-B) / C}] × 100

The results are shown in Table 2 and FIG.

Condition IC ₅₀
Swine Influenza (H1N1)
Avian Influenza (H9N2)
Swine flu
(novel H1N1) Tamiflu
Resistant swine flu
(H1N1 H274Y) Compound 1 (ug / ml) 23.29 ± 2.08 15.46 ± 1.69 11.15 ± 0.52 7.73 ± 0.85 Compound 2 (μg / ml) 23.54 ± 3.68 22.45 ± 3.45 11.54 ± 0.35 13.01 ± 0.41 Compound 3 (μg / ml) 28.42 ± 1.47 25.59 ± 2.14 9.33 ± 0.6 12.8 ± 1.07 Compound 4 (ug / ml) 26.81 ± 2.18 24.77 ± 2.45 13.41 ± 1.09 9.14 ± 0.39 Compound 5 (ug / ml) 26.87 ± 3.81 19.81 ± 3.34 13.68 ± 0.89 10.80 ± 0.48 Tamiflu (ng / ml) 39.74 ± 1.54 4.94 ± 0.56 21.09 ± 1.19 5.13 ± 0.23

When confirming the results of Table 2 and Figure 2, the polygala carenium extract of the present invention and xanthone-based compounds 1 to 5 isolated from it is influenza Avian influenza virus, swine influenza virus, swine flu, Tamiflu resistant swine flu virus Neuraminidase inhibitory activity against was found to be excellent.

Meanwhile, the concentration of 4-methoxyumbelliferone was measured before adding 0.1M glycine-NaOH buffer to stop the reaction for the kinetic study of the compound, and calculated in Sigmaplot 11.0 (SPCC Inc., Chicago, IL). In order to measure the reversible reaction, it was measured by diluting the reaction solution containing the enzyme and the inhibitor. The results are shown in FIG. 3, and it was confirmed that Compound 3 of the present invention inhibited neuraminidase of swine influenza virus as a non-competitive inhibitor (Lineweaver Burk plots of FIG. 3A and FIG. 3B Dixon). see plot).

< Example  7: Cytopathic effect  recovery Assay >

After infection with porcine influenza virus (A / Sw / Kor / CAN1 / 04 (H1N1, KCTC 11165BP) in MDCK (Madin-Darby, canine kidney) cells, the degree of inhibition by virus-treated cell lesions ( Cytopathic effect reduction assay (CPE reduction assay) was performed to evaluate the degree of recovery of the cells (CPE), which shows the morphological changes caused by viral infection and the degeneration of the cell shape. Changes are associated with infection of viruses (Semin. Neurol., 1999, 19, 113-127; J. Virol. Methods, 2002, 106, 71-79).

To this end, first, MDCK cells were dispensed in a 96-well plate at a density of 1 × 10 cells / well to give 5% fetal bovine serum (FBS), 1% penicillin + streptomycin (PS) and L-glutamine (L-). After a day of growth in DMEM culture medium containing glutamine, 2 ml of 500 ml medium), the culture was washed with PBS (phosphate buffer saline) solution. Then, the infection medium: handle (infection media DMEM + 0.5% BSA + 1㎍ / ㎖ trypsin) swine influenza virus, the TCID 50 was inoculated to 100㎕ a concentration of ₅₀ / 100㎕ / well compound 1-5 on and, Cytotoxicity by virus was measured by additional culture for 3 to 5 days.

In order to measure the effect of CPE, MTT experiment was performed to measure the survival of MDCK cells after 5 days of culture. The MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, Sigma M-2128 1G)) reagent is a pale yellow substrate that is cleaved by respiratory enzymes in the mitochondria of living cells and dark blue. Produces a formazan. MTT reactions do not occur in dead cells because of the cytotoxicity of MDCK cells killed by the virus or the substance itself. Therefore, the efficacy of the compound was evaluated using the method of measuring formazan, which produced cytotoxicity of the substance itself and cytotoxicity by virus at absorbance of 550 nm, respectively. That is, the concentration of the compound without MDCK cytotoxicity by the compound was determined, and the difference in degeneration of MDCK cells by the infected swine influenza virus was measured, and the effect of CPE according to the concentration of the compound was shown in FIG. 4.

Referring to FIG. 4, in FIG. 4A, the cell viability was recovered to about 50% in the group treated with the compounds 1 to 5 of 5 μg / ml, and 50% cell death of the compound 3 in FIG. 4B. When the concentration was restored to the compound 3 was found that the cell viability was recovered more than 50% at 4.50 ± 0.14 µg / ml. 4C shows that most of the cells treated with swine influenza virus were abnormal or dead in cell morphology, but the group treated with Compound 3 of the present invention at 5 µg / ml was treated with Tamiflu at 2 µg / ml. It was shown to have cell morphology and viability similar to that of the group, demonstrating the superior antiviral effect of the compounds of the present invention.

< Example  8: Toxicity Test>

Example  8-1. Acute toxicity

This experiment was carried out to investigate the toxicity of the polygala carenium methanol extract and compounds 1 to 5 in acute (within 24 hours) in animals in a short period of time and to determine the lethality. 70 ICR mouse strains, which are common mice, were prepared, and 10 rats were assigned to each group. In the control group, only 30% PEG-400 was administered, and the experimental group was orally administered polygala carenium methanol extract and compounds 1 to 5 at a concentration of 1.0 g / kg. The mortality was examined 24 hours after administration, and all of them survived in the control group, the experimental group administered the polygala carenium methanol extract and the compounds 1 to 5 at 1.0 g / kg concentration.

Example  8-2: Experimental group  And control organ organs and tissue toxicity experiments

In the long-term toxicity experiment, C57BL / 6J mice were administered with only one experimental group and a solvent in which Compound 1-5 and methanol extract were administered once at a concentration of 1.0 g / kg in order to investigate the effect on each organ (tissue) of the animal. Blood was collected after 8 weeks from animals in the control group and the blood concentrations of glutamate-pyruvate transferase (GPT) and blood urea nitrogen (BUN) were measured using a Select E (Vital Scientific NV, Netherland) instrument. As a result, GPT, which is known to be related to hepatotoxicity, and BUN, which is known to be related to renal toxicity, showed no significant difference compared to the control group. In addition, liver and kidneys were excised from each animal, and histological observations were performed by optical microscopy through a conventional tissue fabrication process. No abnormalities were observed.

< Examples  1: Pharmaceutical Formulation example >

1-1. Manufacture of tablets

200 g of polygala carenium methanol extract of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. To this mixture was added a 10% gelatin solution, which was pulverized and passed through a 14-mesh sieve. This was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into tablets.

1-2. Injection preparation

1 g of the polygalar carenium methanol extract 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 DEG C for 30 minutes.

< Examples  2: Food Manufacturing example >

2-1. Manufacture of cooking seasonings

Polygala calenium methanol extract of the present invention was added to the cooking seasoning at 0.2 to 10% by weight to prepare a cooking seasoning for health promotion.

2-2. Manufacture of flour food products

Polygalar carenium methanol extract of the present invention was added to the flour in 0.1 to 5.0% by weight, and using this mixture to prepare bread, cakes, cookies, crackers and noodles to prepare health promoting food.

2-3. soup  And juicy ( gravies Manufacturing

Polygala carenium methanol extract of the present invention was added to the soup and juice in 0.1 to 1.0% by weight to prepare meat products for health promotion, soup and noodles of noodles.

2-4. dairy product( dairy products Manufacturing

Polygalar carenium methanol extract of the present invention was added to the milk in 0.1 to 1.0% by weight, using the milk to prepare a variety of dairy products such as butter and ice cream.

< Examples  3: Drink Manufacturing example >

3-1. Vegetable juice  Produce

0.5 g of polygala carenium methanol extract of the present invention was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice for health promotion.

3-2. Fruit juice  Produce

0.1 g of polygala carenium methanol extract of the present invention was added to 1,000 ml of apple or grape juice to prepare fruit juice for health promotion.

< Examples  4: Feed additives Manufacturing example >

A feed additive was prepared by mixing 10.0 g of polygalar carenium methanol extract of the present invention with 50.0 g of feed excipient. However, the compounding ratio may be arbitrarily modified, and the above components may be mixed and used in the manufacture according to a conventional feed composition manufacturing method.

< Examples  5: Disinfectant Manufacturing example >

After adding 3.0 g of the polygalar carenium methanol extract of the present invention or the xanthone-based compound separated therefrom, purified water was added to 1,000 ml to prepare a natural antiseptic for antiseptic.

Claims (8)

1,3-dihydroxyxanthone (Compound 1 ), 1,7-dihydroxyxanthone (1,7-dihydroxyxanthone, Compound 2 ), 1,7-dihydroxy- 4-methoxyxanthone (1,7-dihydroxy-4-methoxyxanthone, compound 3 ), 1,3,7-trihydroxyxanthone (1,3,7-trihydroxyxanthone, compound 4 ), and 1,2,3 1,2,3,5-tetrahydroxyxanthone (compound 5 ), containing at least one xanthene compound selected from the group consisting of Composition for the prevention or treatment of colds, avian influenza, swine influenza or swine flu containing Polygala karensium extract.
[Formula 1]

The method of claim 1,
The polygala calenium extract is a cold, avian influenza, swine influenza or swine flu, characterized in that the extract is prepared by extraction with water, lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, chloroform or a mixed solvent thereof. Or a therapeutic composition. delete 1,3-dihydroxyxanthone (Compound 1 ), 1,7-dihydroxyxanthone (1,7-dihydroxyxanthone, Compound 2 ), 1,7-dihydroxy- 4-methoxyxanthone (1,7-dihydroxy-4-methoxyxanthone, compound 3 ), 1,3,7-trihydroxyxanthone (1,3,7-trihydroxyxanthone, compound 4 ), and 1,2,3 1,2,3,5-tetrahydroxyxanthone (compound 5 ), containing at least one xanthene compound selected from the group consisting of A dietary supplement for the prevention or amelioration of colds, avian influenza, swine influenza or swine flu, containing Polygala karensium extract.
[Formula 1]

5. The method of claim 4,
The polygala calenium extract is a cold, avian influenza, swine influenza or swine flu, characterized in that the extract is prepared by extraction with water, lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, chloroform or a mixed solvent thereof. Or dietary supplements for improvement. 1,3-dihydroxyxanthone (Compound 1 ), 1,7-dihydroxyxanthone (1,7-dihydroxyxanthone, Compound 2 ), 1,7-dihydroxy- 4-methoxyxanthone (1,7-dihydroxy-4-methoxyxanthone, compound 3 ), 1,3,7-trihydroxyxanthone (1,3,7-trihydroxyxanthone, compound 4 ), and 1,2,3 1,2,3,5-tetrahydroxyxanthone (compound 5 ), containing at least one xanthene compound selected from the group consisting of Polygala ( Polygala) Animal medicine for the prevention or treatment of cold, avian influenza, swine influenza or swine flu containing karensium ) extract.
[Formula 1]

1,3-dihydroxyxanthone (Compound 1 ), 1,7-dihydroxyxanthone (1,7-dihydroxyxanthone, Compound 2 ), 1,7-dihydroxy- 4-methoxyxanthone (1,7-dihydroxy-4-methoxyxanthone, compound 3 ), 1,3,7-trihydroxyxanthone (1,3,7-trihydroxyxanthone, compound 4 ), and 1,2,3 1,2,3,5-tetrahydroxyxanthone (compound 5 ), containing at least one xanthene compound selected from the group consisting of Polygala ( Polygala) Animal feed additive for the prevention or amelioration of cold, avian influenza, swine influenza or swine flu containing karensium ) extract.
[Formula 1]

1,3-dihydroxyxanthone (Compound 1 ), 1,7-dihydroxyxanthone (1,7-dihydroxyxanthone, Compound 2 ), 1,7-dihydroxy- 4-methoxyxanthone (1,7-dihydroxy-4-methoxyxanthone, compound 3 ), 1,3,7-trihydroxyxanthone (1,3,7-trihydroxyxanthone, compound 4 ), and 1,2,3 1,2,3,5-tetrahydroxyxanthone (compound 5 ), containing at least one xanthene compound selected from the group consisting of Polygala ( Polygala) karensium ) A disinfectant for the prevention of colds, avian influenza, swine influenza or swine flu.
[Formula 1]

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