KR20140023357A - Pandemic influenza antiviral agent - Google Patents

Abstract

An anti-influenza virus agent comprising an extract obtained by extracting a Chinese quince with 50% ethanol water and column extracting and purifying the extract as an active ingredient.

Description

Anti-influenza virus {PANDEMIC INFLUENZA ANTIVIRAL AGENT}

The present invention relates to an anti-influenza virus agent, a novel influenza virus hemagglutination inhibitor, and an adsorption inhibitor against cells of the new influenza virus, comprising as an active ingredient an extract derived from a plant having an infection inhibitory effect of the new influenza virus.

The influenza virus that causes the pandemic of influenza every year is an RNA virus having an envelope membrane on the order of 10,000 millimeters in diameter. It is classified into three types of A, B, and C from the difference in antigenicity, but it is A type and B type which show fashionable spread. On the particle surface of these viruses, two types of glycoproteins, erythrocyte agglutinates (HA) and neuraminidase (NA), protrude in the form of spikes, and there are eight segmented gene RNAs inside. HA and NA on the surface of the virus frequently cause mutations within the same subtype, resulting in new antigenic variants each year.

Influenza viruses released by cough droplets invade the human nose or mouth and are adsorbed to the mucosal epithelial cells of the upper respiratory tract by spiked glycoprotein HA on the surface of the virus and begin proliferation after invasion into the cells. Recent research has revealed the infection mechanism of viruses. The virus binds to receptors consisting of oligosaccharides present on the surface of human target cells, is taken in by the endosome, invades into the cell by fusion of the viral membrane and the endosome membrane, passes through the shelling and migration, Replication takes place and finally progeny virus particles are formed and propagated by budding from the host cell membrane.

Influenza virus infection causes symptoms such as sudden fever, headache, joint pain and general boredom in a few days, and before and after it, respiratory symptoms such as cough, sore throat, runny nose and stuffy nose appear. Unlike the so-called cold, it is characterized by strong infectivity that causes an explosion in the short term. In addition, the structure of the HA protein of influenza virus is mutated every year, and the fact that antibodies produced by past infections are not so helpful is a factor in spreading the infection.

In order to suppress the infection of the influenza virus, inhibition of the adsorption to epithelial cells, inhibition of invasion to cells, inhibition of gene transcription and replication, inhibition of protein synthesis, inhibition of release from cells, and the like can be considered. It is the target of this antiviral drug. To date, antiviral drugs such as amantadine, rimantadine, and zanamivir have been developed, but side effects such as hypersensitivity, mental neurological symptoms, digestive system symptoms, and autonomic nervous system symptoms have been reported, and caution is required for their application. Do.

Moreover, influenza virus can be considered to be difficult to suppress infection by vaccination since the infection and proliferation in the airway mucosal epithelium or the epidemic of the year cannot be accurately predicted. Avoiding frequent gargles and dry throats, getting plenty of nutrition and rest can be considered the most effective precautions at present. There is a demand for the development of anti-influenza virus agents that have high infection inhibitory effects, have no safety problems, and which can be used on a daily basis.

In recent years, polyphenol components of tea and black tea have been reported as anti-influenza materials derived from natural products (Non-Patent Documents 1 and 2), and it has been found that goggle of black tea suppresses the actual viral infection by a test using human ( Non-Patent Document 3). In addition, it has been reported that the flavonoid component derived from gold has an influenza infection inhibitory effect by the sialidase inhibitory activity of the virus (Non-Patent Document 4). In addition, Gyejiyiwolbiiltang (patent document 1), black gooseberry extract (patent document 2), potato anthocyanin pigment (patent document 3), guava leaf extract (patent document 4), napoma extract (patent) Document 5) and antiviral effects, such as olive leaf extract (patent document 7), are reported.

Moreover, in a rose family, the anti-influenza agent which uses the extract of the bud or the petal as an active ingredient is disclosed (patent document 6), the extract of a Chinese quince (patent document 8), and the column fraction of a quince (patent document 9, non The anti-influenza virus effect of Patent Document 5), the inhibitory effect of the quince fraction by the plaque test method against the new influenza virus (Non Patent Literature 6), and the extract of the quince inhibits aggregation of erythrocytes by influenza virus (N Patent document 7) is reported.

Chinese quince (Chaenomeles sinensis) is a deciduous tree native to China, and the fruit part is edible as Chinese quince, pickled sugar, syrup and the like. The fruit is called Meisa and is prescribed as a herbal medicine with expectoration, Jinhae, and pain relief. The active ingredients in Chinese quince fruit have been identified, and triterpenes such as oleanoic acid have been reported as anti-inflammatory components and high-molecular polyphenols as antibacterial components against hemolytic streptococcus pyogenes, the causative agent of pharyngitis. The present inventors have reported the effect of inhibiting hemagglutination and infection of the macromolecular polyphenols in seasonal fruit against the seasonal influenza virus (Patent Document 9).

However, reports on the effect of inhibiting the aggregation of erythrocytes by the new influenza virus and the effect of inhibiting the adsorption of the new influenza virus to cells by the column fractions of the quince disclosed in the present invention patent have not been shown, and were first disclosed by the present invention. will be. Influenza repeats the epidemic every year, sometimes causing a worldwide pandemic of fenders. Influenza caused by the new H1N1 virus, which originated in Mexico in March 2009, is one of them. This time, the quinocyte extract inhibited the erythrocyte aggregation inhibitory effect and the infection inhibitory effect against the new influenza virus A / Chiba / 1001/2009 (H1N1) pdm.

Japanese Unexamined Patent Publication No. 6-199680 Japanese Laid-Open Patent Publication 2000-212092 Japanese Unexamined Patent Publication No. 2001-316399 Japanese Laid-Open Patent Publication 2000-273048 Japanese Patent Application Laid-Open No. 11-71296 Japanese Unexamined Patent Publication No. 2002-145790 Japanese Patent Publication No. 2002-020305 Japanese Patent Application Laid-Open No. 2005-343836 PCT / JP2010 / 005762 Specifications

 Infectious Disease Magazine, 68 (7) 824-829 (1994)  Infectious Disease Magazine, 70 (11) 1190-1192 (1996)  Infectious Disease Magazine, 71 (6) 487-494 (1997)  Chem. pharm. Bull. 38 (5) 1329-1332 (1990)  Journal of Ethnopharmacology, 118, 108-112 (2008)  Food News, 43 (1) 155-157 (2009)  J Agric. Food Chem. 53, 928-934 (2005)

It is an object of the present invention to provide a novel influenza virus infection inhibitor, an inhibitor of red blood cell aggregation by the new influenza virus, and an adsorption inhibitor against cells of the new influenza virus, using a safe plant extract that can be used with confidence in daily life. .

In order to solve the above problems, the present inventors pay attention to quince without high side effects and high safety, and extracts of quince in MDCK cells to which new influenza virus is added, that is, a fraction containing abundant polyphenols in quince fruit (CSD 3) The novel influenza virus A / Chiba / 1001/2009 (H1N1) pdm was found to have an infectious neutralizing activity and an effect of inhibiting erythrocyte aggregation of the quinine fraction (CSD 3) against the new influenza virus, thereby completing the present invention.

In other words, the present invention is characterized in that the extract of Chinese quince (Chaenomeles sinensis, Pseudocydonia sinensis) as an active ingredient, the new influenza virus infection inhibitor, the hemagglutination inhibitor in the new influenza virus infected cells, the adsorption of cells to the new influenza virus Inhibitor. In addition, the present invention is a food and drink having an anti-new influenza virus action containing a quince extract.

SUMMARY OF THE INVENTION The present invention provides extracts of high safety quince as active ingredients, and provide an influenza virus infection inhibitor that is strong against new influenza virus, an inhibitor of hemagglutination in cells infected with influenza virus, and an adsorption inhibitor against cells of new influenza virus. . In addition, the extract of the Chinese quince, which is an active ingredient of the present invention, has high safety and can be widely used in daily life as a novel influenza virus infection suppressing product by adsorption, impregnation, and addition to masks, air conditioner filters, clothes, wet tissues, and spray liquids. have. Furthermore, it can be added to foods, such as chewing gum, candy, tablets, and beverages, and can be used and consumed routinely as a food which has anti-influenza virus action. The present invention is effective for preventing infection of the new influenza virus and for treating diseases caused by the new influenza virus.

1 is a graph showing the erythrocyte aggregation inhibitory effect of the Chinese quince extract CSD 3.
Fig. 2 is a graph showing the A / Chiba / 1001/2009 (H1N1) pdm infectious neutralizing activity (plaque method) of the Chinese quince extract CSD 3.
3 is a graph showing the A / Chiba / 1001/2009 (H1N1) pdm infectious neutralizing activity (TCID ₅₀ method) of the Chinese quince extract CSD 3.

In the Chinese quince which becomes a raw material of this invention, it is preferable to use the fruit.

The method for obtaining the extract of the present invention from the milled product of the plant is not particularly limited, but lower alcohols such as water, methanol, ethanol, n-propanol and n-butanol, ether, ethyl acetate, acetone, glycerin, propylene glycol and the like 1 type, or 2 or more types of mixed solvent of the organic solvent of is added, and it extracts by the extraction method conventionally performed. However, in consideration of oral ingestion of the anti-influenza virus agent of the present invention, it is preferable to extract using water, ethanol or a mixture thereof in terms of safety.

There is no restriction | limiting in particular as extraction conditions, About 1 to 5 hours are preferable at 50-90 degreeC. The extract is filtered and the extractant is distilled off, followed by concentration or freeze drying under reduced pressure. Moreover, what extracted these extracts by the organic solvent fraction, column chromatography, etc. can also be used.

There is no restriction | limiting in particular about the usage form of this invention, A powder, a tablet, a troche, an inhalant, a gargle drug, is added to a plant extract illustrated as an active ingredient by adding a solvent, a dispersing agent, a preparation carrier, an emulsifier, a diluent, a stabilizer, etc. It is possible to prepare as any agent such as a water-soluble agent, suppository, or injection, and examples of the route of administration include oral administration, airway administration, intravenous administration, rectal administration, subcutaneous administration, intradermal administration, and the like. In this case, the dosage for adults is preferably 10 to 2000 mg / day in each extract, but is not limited to this value. The amount of the extract added to the various formulations varies depending on the form of the formulation, but is preferably added in an amount of at least 0.001% by weight, preferably at least about 0.01% by weight.

In addition, the present invention can provide an infection suppressing article that can contribute to the prevention of influenza by adsorbing, impregnating, and adding a mask, an air conditioner filter, clothing, a wet tissue, and a spray solution. Although the adsorption and addition amount of the plant extract in these uses differs according to the form of the infection suppressing article, it cannot be uniformly prescribed | regulated, but it is preferable to add so that it may become a ratio of 0.001-5 weight%.

In addition, the present invention is high in safety, and is compounded into foods such as sweets such as chewing gum, candy, sweets, gummi jelly, chocolate and biscuits, ice cream and sorbets such as sherbets, beverages, soups and jams. Can be used as The amount added is different depending on the form of use and the fineness of the extract, but it is preferably added in an amount of 0.001 to 5% by weight, preferably about 0.01 to 1% by weight relative to the food and drink.

Example

Hereinafter, the present invention will be described in detail with reference to Examples and Test Examples, but the present invention is not limited thereto.

Example 1

Preparation of Cell Lines and Viruses

Cells were cultured in Eagle's minimum essential medium (EMEM) containing 10% calf serum using Mardin-Darby canine kidney (MDCK) cells. Influenza viruses used A / Udorn / 307/72 (H3N2) and A / Chiba / 1001/2009 (H1N1) pdm. A / Udorn / 307/72 was inoculated and propagated in embryonated eggs (11 eggs) in the intestinal lumen cavity, and was subjected to Temperature-Sensitive Mutants of Influenza A / Udorn / 72 (H3N2) Virus. Purification by the method described in Virology 117, 45-61 (1982). The A / Chiba / 1001/2009 (H1N1) pdm strain was inoculated into MDCK cells and grown for 2 days in a carbon dioxide gas incubator (37 ° C., 5% CO ₂ ). It was centrifuged at 5,000 rpm for 5 minutes, and the supernatant liquid was made into the virus liquid.

Example 2

Purification of the active ingredient in Chinese quince

The dried fruits of C. sinensis Koehne were obtained from the market of Hubei Province of China. 100 g of the dried fruit was refluxed with 50% ethanol (700 mL) for 1 hour, and after filtration, the extract was concentrated under reduced pressure and freeze-dried to obtain a 50% ethanol extract (CSE50) of the Chinese quince (23 g). The CSE50 was suspended with water, and five steps of hydrous ethanol (0%, 20%, 40%, 60%, 100% ethanol) by Diaion HP-20 (Mitsubishi Chemical) column chromatography (column: 8 x 15 cm). ), CSD 1 (water eluting fraction: 14.5 g), CSD 2 (20% ethanol eluting fraction: 3.2 g), CSD 3 (40% ethanol eluting fraction: 3.7 g), CSD 4 (60% ethanol eluting) Fractions: 161 mg) and five fractions of CSD 5 (100% ethanol eluting fractions: 161 mg) were obtained. Condensed polyphenols in each fraction were quantified by vanillin hydrochloric acid method using (-)-epicatechin as a standard.

Example 3

Determination of Polyphenol Content and Infectious Neutralization Value of Each Chinese Quince Fraction

Infection neutralization value of each fraction was measured by the plaque method using A / Udorn / 307/72 (H3N2). To the 0.1 ml virus solution (1,000 PFU / ml), an equal amount of the fraction 10-fold dilution was added, reacted for 30 minutes at room temperature, 100 µl was inoculated into MDCK cells (6 well plate), and adsorbed at room temperature for 60 minutes. . After adsorption, 1.6 ml of Leibovitz's L-15 medium (Life Technologies Japan Ltd.) containing 0.6% agarose, 1.5% gelatin and 2.5 μg / ml trypsin was added and solidified. After incubation for 3 days at 34 ° C, the number of plaques that appeared was measured to determine 50% infection neutralization concentration (IC ₅₀ ). TGS (25 mM Tris, 140 mM sodium chloride, 5 mM potassium chloride, 0.7 mM sodium phosphate 12 hydrate, 5.6 mM glucose, pH 7.4) was used for dilution of the virus and fractions.

As in Example 2, 50% ethanol extract of the Chinese quince was eluted with 5 steps of hydrous ethanol using DiaionHP-20, and fractionated to A / Udorn / 307/72 (H3N2) of each fraction fractionated by CSD 1-5. The 50% infection neutralizing IC ₅₀ and polyphenol content for each is shown in Table 1. Three fractions of CSD had the highest infection inhibitory effect (IC ₅₀ = 0.8 µg / ml). In addition, CSD 3 had the highest polyphenol content of 53.8%.

Therefore, in subsequent experiments, it was assumed that the antiviral effect against the new influenza virus A / Chiba / 1001/2009 (H1N1) pdm was determined using CSD 3.

a; 50% infection neutralization concentration for A / Udorn / 307/72

b; Epicatechin equivalent, n = 3, mean ± standard deviation

Example 4

Erythrocyte attenuation reduction test for new influenza virus A / Chiba / 1001/2009 (H1N1) pdm of CSD 3 treatment

Erythrocyte agglutination value (HA value) was measured using a 96 well plate. Phosphate buffered saline was used to make a 2-fold dilution series (50 μl) of CSD 3 treated virus at each concentration, 50 μl of 0.5% (v / v) chicken erythrocytes were added, and stopped at 4 ° C. for 1 hour. The presence or absence of erythrocyte aggregation was determined, and the reciprocal of the highest dilution showing aggregation was taken as the HA of each virus solution. The inhibitory effect of erythrocyte aggregation on the new influenza virus A / Chiba / 1001/2009 (H1N1) pdm of quince CSD 3 fractions was evaluated by HA reduction test. Equivalent amount of CSD 3 solution (0: control, 0.1, 2, 10, 50, 500 µg / ml) was added to 0.1 ml of virus solution (HA value: 32), and the HA value was measured after reacting at room temperature for 60 minutes. .

According to the above method, the erythrocyte aggregation inhibitory effect of A / Chiba / 1001/2009 (H1N1) pdm by CSD 3 treatment was evaluated by the erythrocyte aggregation reduction test. The hemagglutination test is based on the reaction of hemagglutinin on the surface of the virus with the receptors of the red blood cells, and is a test for evaluating the receptor binding ability of the virus. In CSD 3 of 50 µg / ml or more, the hemagglutination effect of CSD 3 itself was observed. Therefore, the decrease in hemagglutination of A / Chiba / 1001/2009 (H1N1) pdm was tested using CSD 3 of 25 µg / ml or less. Was carried out. At 1 µg / ml or less, the HA value of the virus was 16, the same as that of the untreated virus, and no decrease in the hemagglutination value was observed. In the treatment of 5 µg / ml, it was reduced to 1/2 as 8. At 25 μg / ml it was reduced below the detection limit (2) (FIG. 1). From the above results, the effect of inhibiting erythrocyte aggregation by the new influenza of the Chinese quince fraction and the effect of inhibiting the adsorption of new influenza virus to the cells were recognized.

Example 5

Reduction of CSD 3 Infection with New Influenza Virus A / Chiba / 1001/2009 (H1N1) pdm

Infectious neutralization effect against new influenza virus A / Chiba / 1001/2009 (H1N1) pdm of quince CSD 3 fractions was evaluated by infection reduction test. Equivalent amount of CSD 3 solution (0: control, 0.1, 2, 10, 50, 500 μg / ml) was added to 0.1 ml of virus solution (2 × 10 ⁷ PFU / ml, HA value: 32) for 60 minutes at room temperature. The infection was measured by plaque method and Tissue Culture Infectious Dose 50% (TCID ₅₀ ) method.

i) measurement by plaque method

A series of 10-fold dilutions of CSD 3 treated virus at each concentration were made, 100 μl were inoculated into MDCK cells (6 well plates) and plaques were counted by the method described above. Infected values are expressed as plaque forming units (PFU) / ml.

Ii) measurement by TCID ₅₀ method

A series of 10-fold dilutions of CSD 3 treated virus at each concentration were made of MEM containing 2.5 μg / ml trypsin, 200 μl were inoculated into MDCK cells (24 well plates) and incubated for 3 days at 37 ° C. in a carbon dioxide incubator. . After incubation, infection was determined by the presence or absence of hemagglutination activity in the supernatant, and the amount of TCID ₅₀ was determined.

According to the above method, infection neutralizing activity was evaluated by the test for reducing infection by the plaque method. The infection value of the untreated virus was 1.0 × 10 ⁷ PFU / ml. Inhibition of infection was confirmed by CSD 3 in a dose-dependent manner, and the infection was reduced to about 2/3 at 1 μg / ml, about 1/60 at 25 μg / ml, and about 1 / 10,000 at 250 μg / ml. (FIG. 2). However, since there were many microplaques and accurate measurement was difficult, the infection by TCID ₅₀ method was evaluated by the reduction test.

As a result of measurement by TCID ₅₀ method, about 1/3 of CSD 3 treatment at 1 µg / ml, about 1/10 at 5 µg / ml, about 1/100 at 25 µg / ml, and 1 / 3,000 at 250 µg / ml Infection was inhibited (FIG. 2). In addition, at the same time as the evaluation of the TCID _50, cytopathic effect under the microscope: when evaluating the TCID ₅₀ by the observation of (Cytopathic effect CPE), the results were exactly the same.

In the 5 µg / ml CSD 3 treatment, the decrease in hemagglutination was about 1/2, while the decrease in infection was about 1/10. The decrease in infectivity was five times that of erythrocyte agglutination, suggesting that CSD 3 mainly inhibits the virus proliferation process after adsorption of the new influenza virus A / Chiba / 1001/2009 (H1N1) pdm.

From the above results, when the activity fraction CSD 3 of the Chinese quince was evaluated, the hemagglutination inhibitory activity and the infectious neutralizing activity of the new influenza virus A / Chiba / 1001/2009 (H1N1) pdm were evaluated at 5 µg / ml. The virus treated in 3 was found to have a erythrocyte agglutination value of about 1/2, which reduced the infectivity to about 1/10. Treatment at 250 μg / ml reduced the infectivity to 1 / 3,000. These results show that the anti-influenza virus active ingredient in the Chinese quince is effective against the H1N1 novel influenza virus. In addition, the presence of an inhibitory effect after the virus adsorption step was suggested in that the infectivity was reduced beyond the decrease in the hemagglutination.

Using the Chinese quince extract prepared in Example 1, gargle, inhalant, troche, spray, chewing gum, candy, fruit, beverage, powder, tablet, water, gummi jelly, chocolate, biscuit, ice, sherbet, Soup, jam, wet tissue, and mask were prepared. The prescription is shown below as an example.

Example 6

Prescription of gargle

Ethanol 2.0 wt%

1.0% by weight fragrance

Saccharin 0.05 wt%

0.01% by weight of chlorhexidine hydrochloride

CSD 3 0.5 wt%

Water rest

                        100.0 wt%

Example 7

Prescription of inhalants

Ethanol 5.0 wt%

CSD 3 1.0 wt%

Water rest

                         100.0 wt%

Example 8

Prescription of Trocheze

Glucose 72.3 wt%

Lactose 19.0 wt%

Arabian rubber 6.0 wt%

1.0% by weight fragrance

Sodium monofluorophosphate 0.7 wt%

CSD 3 1.0 wt%

                          100.0 wt%

Example 9

Prescription of Spray

Ethanol 25.0 wt%

Citric acid 1.5 wt%

Trisodium citrate 1.0 wt%

CSD 3 0.5 wt%

Water rest

                          100.0 wt%

Example 10

Prescription of Choung Sword

Gum base 20.0 wt%

54.7 wt% sugar

15.0% by weight glucose

Starch syrup 9.3 wt%

0.5% by weight fragrance

CSD 3 0.2 wt%

                          100.0 wt%

Example 11

Prescription of Candy

50.0 wt% sugar

Starch syrup 34.0 wt%

Citric Acid 1.0 wt%

Fragrance 0.2 wt%

CSD 3 0.4 wt%

Water rest

                         100.0 wt%

Example 12

Prescription

Sugar 76.1 wt%

Glucose 19.0 wt%

0.2 wt% sucrose fatty acid ester

Fragrance 0.2 wt%

CSD 3 0.5 wt%

Water rest

                          100.0 wt%

Example 13

Prescription of beverages

Orange Juice 30.00 wt%

15.24% by weight per isomerization

Citric Acid 0.10 wt%

Vitamin C 0.04 wt%

Perfume 0.10 wt%

CSD 3 0.10 wt%

Water rest

                         100.00 wt%

Example 14

Prescription of Powder

Corn starch 55.0 wt%

Carboxycellulose 40.0 wt%

CSD 3 5.0 wt%

                         100.0 wt%

Example 15

Prescription of tablets

Lactose 70.0 wt%

15.0% by weight of crystalline cellulose

Magnesium Stearate 5.0 wt%

CSD 3 10.0 wt%

                          100.0 wt%

Example 16

Prescription of Hydrant

Ethanol 2.00 wt%

1.00% by weight

Saccharin 0.05 wt%

0.01% by weight of chlorhexidine hydrochloride

CSD 3 0.50 wt%

Water rest

                        100.00 wt%

Example 17

Prescription of Gumi Jelly

Gelatin 60.00 wt%

Starch syrup 23.00 wt%

8.50 wt% sugar

4.50 wt% plant fat

Mannitol 2.95 wt%

Lemon Juice 1.00%

CSD 3 0.05 wt%

                         100.00 wt%

Industrial availability

INDUSTRIAL APPLICABILITY The present invention can be widely used in daily life as a novel influenza virus infection suppressing article by adsorption, impregnation, and addition to masks, air conditioner filters, clothes, wet tissues, and spray liquids. In addition, it can be added to various foods and used and consumed daily as foods having an anti-new influenza virus action. The present invention is effective for preventing infection of the new influenza virus and for treating diseases caused by the new influenza virus. That is, as an infection prevention material, the application as a material of new product development can be expected.

This application claims the priority in Japanese Patent Application No. 2011-098230 for which it applied on April 26, 2011, and quotes the content as a part of this application.

Claims (5)

An anti-influenza virus agent comprising an extract of Chinese quince (Chaenomeles sinensis, Pseudocydonia sinensis) as an active ingredient. An anti-influenza virus agent comprising an extract obtained by extracting a Chinese quince with 50% ethanol water and column extracting and purifying the extract as an active ingredient. An inhibitor of aggregation of erythrocytes by a new influenza virus, comprising extracts obtained by extracting a Chinese quince with 50% ethanol water, and extracting the extract by column fractionation and purifying the extract. An inhibitor of adsorption to a new influenza virus cell comprising an extract obtained by extracting a Chinese quince with 50% ethanol water and subjecting the extract to column fraction purification. Food and drink with anti-new influenza virus action containing Chinese quince extract.

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