JP2012229178A - Pandemic influenza antiviral agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a pandemic influenza antiviral agent having an infection-inhibiting action on an influenza virus.SOLUTION: A pandemic influenza antiviral agent contains an extract obtained by extracting Chinese quince using 50% ethanol-water and purifying the extract solution by column fractionation, as an active ingredient.

Description

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

  The influenza virus that causes the influenza epidemic is an RNA virus having an envelope membrane with a diameter of about 1 / 10,000 mm in diameter every year. Although it is classified into three types, A, B, and C, due to the difference in antigenicity, it is A type and B type that show a trendy spread. Two types of glycoproteins, hemagglutinin (HA) and neuraminidase (NA), protrude in the form of spikes on the surface of these virus particles, and gene RNA segmented into 8 segments exists inside. HA and NA on the surface of the virus frequently mutate within the same subtype, and new antigenic variants appear every year.

  Influenza virus released by droplets due to cough enters the human nose and mouth, adsorbs to the mucosal epithelial cells of the upper respiratory tract by the spike-like glycoprotein HA on the surface of the virus, and begins to proliferate after entering the cells. Recent research has revealed the mechanism of viral infection. The virus binds to a receptor consisting of a sugar chain on the surface layer of human target cells, is taken into the endosome, enters the cell by fusion of the viral membrane and endosomal membrane, undergoes shelling and translocation, and the expression of the viral gene Replication occurs, and finally progeny virus particles are formed and propagated by budding from the host cell membrane.

  Infection of influenza virus causes symptoms such as sudden fever, headache, joint pain, general malaise in a few days, followed by respiratory symptoms such as cough, sore throat, runny nose and stuffy nose. Unlike the so-called cold, it is highly infectious and causes an explosive epidemic in a short period of time. In addition, the structure of the HA protein of influenza virus repeats mutations every year, and the fact that antibodies produced by past infections are not very useful is also a factor that spreads infection.

  In order to suppress infection with influenza virus, inhibition of adsorption to epithelial cells, inhibition of entry into cells, inhibition of gene transcription / replication, inhibition of protein synthesis, inhibition of release from cells, etc. are considered. Each is a target for antiviral drugs. To date, antiviral drugs such as amantadine, rimantadine, and zanamivir have been developed, but side effects such as hypersensitivity, neuropsychiatric symptoms, digestive system symptoms, and autonomic nervous system symptoms have been reported. Caution must be taken.

  In addition, influenza viruses are thought to be difficult to control by vaccination because influenza viruses infect and propagate in the respiratory mucosal epithelium and the epidemic type of the year cannot be accurately predicted. Frequent gargling, avoiding throat dryness, and adequate nutrition and rest are currently considered the most effective preventive measures. Development of an anti-influenza virus agent that is highly effective in suppressing infection and has no safety problems and can be used on a daily basis is desired.

  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 become clear that gargle of black tea suppresses actual viral infections through tests using humans. (Non-Patent Document 3). In addition, it has been reported that ougon-derived flavonoid components exhibit an influenza infection suppression effect due to viral sialidase inhibitory activity (Non-patent Document 4). Furthermore, Kameda Futoshi Koshiketsu (Patent Document 1), Kuroburi Currant Extract (Patent Document 2), Potato Anthocyanin Pigment (Patent Document 3), Guava Leaf Extract (Patent Document 4), Rafu Hemp Antiviral effects such as an extract (Patent Document 5) and an olive leaf extract (Patent Document 7) have been reported.

  Moreover, in the Rosaceae plant, an anti-influenza agent containing an extract of the flower bud or petal as an active ingredient is disclosed (Patent Document 6), an extract of Karin (Patent Document 8), and a column fraction of Karin. (Patent Document 9, Non-Patent Document 5) Anti-influenza virus effect, infection-inhibiting effect of Karin fraction by new plaque test method against new influenza virus (Non-Patent Document 6), and further, Karin extract is agglutination of erythrocytes by influenza virus (Non-patent Document 7) has been reported.

  Karin (Chaenomeles sinensis) is a deciduous tree native to China, and the fruit part is eaten as karin liquor, candied sugar, syrup and the like. The fruit is called Meisa and is prescribed as a Chinese medicine with medicinal properties such as expectorant, antitussive, and analgesic. We identified medicinal ingredients in karin fruits and reported triterpenes such as oleanolic acid as antibacterial components against hemolytic streptococci (Streptococcus pyogenes) that cause pharyngitis, and polymer polyphenols as anti-inflammatory components. ing. The present inventors have reported the hemagglutination suppression effect and the infection suppression effect with respect to seasonal influenza virus of the high molecular polyphenols in a karin fruit (patent document 9).

  However, there has been no report regarding the effect of the column fraction of Karin shown in the patent of the present invention on the aggregation of erythrocytes by the new influenza virus and the effect of suppressing the adsorption of the new influenza virus to cells, and this is the first clear by the present invention. It has been made. Influenza repeats an epidemic every year, causing a global pandemic, sometimes called a pandemic. One example is influenza caused by a new H1N1 virus that originated in Mexico in March 2009. In this study, karin extract was found to have haemagglutination-inhibiting and infection-inhibiting effects against new influenza virus A / Chiba / 1001/2009 (H1N1) pdm.

JP-A-6-199680 JP 2000-212092 A JP 2001-316399 A JP 2000-273048 A JP-A-11-71296 JP 2002-145790 A Special table 2002-020305 gazette JP 2005-343836 A PCT / JP2010 / 005762 specification

Journal of Infectious Diseases, 68 (7) 824-829 (1994) Journal of Infectious Diseases, 70 (11) 1190-1192 (1996) Journal of Infectious Diseases, 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)

  The object of the present invention is to use a highly safe plant extract that can be used with peace of mind on a daily basis, a novel influenza virus infection inhibitor, an agglutination inhibitor of erythrocytes by the novel influenza virus, and an inhibition of adsorption of the novel influenza virus to cells. Is to provide an agent.

  In order to solve the above-mentioned problems, the present inventors focused on Karin which has no side effects and is highly safe, and an extract of Karin in MDCK cells to which a new influenza virus is added, that is, an image rich in polyphenols in Karin fruit. (CSD3) found infectious neutralizing activity against the new influenza virus A / Chiba / 1001/2009 (H1N1) pdm and the hemagglutination-inhibiting effect of the Karin fraction (CSD3) against the new influenza virus. Completed the invention.

  That is, the present invention relates to a novel influenza virus infection inhibitor characterized by containing an extract of karin (Chaenomeles sinensis, Pseudocydonia sinensis), a hemagglutination inhibitor in a novel influenza virus-infected cell, and a novel influenza virus cell It is an adsorption inhibitor. Furthermore, this invention is the food / beverage products which have an anti- new influenza virus effect | action containing a karin extract.

  The present invention comprises a highly safe extract of karin as an active ingredient, an influenza virus infection inhibitor that is strong against new influenza viruses, an hemagglutination inhibitor in influenza virus-infected cells, and a novel influenza virus adsorption inhibitor to cells. It is to provide. In addition, since the extract of Karin, which is an active ingredient of the present invention, has high safety, it is adsorbed, impregnated and added to a mask, air conditioner filter, clothing, wet tissue, spray solution, etc. Can be widely used in daily life. Furthermore, it can be added to foods and drinks such as chewing gum, candy, tablet confectionery, beverages, etc., and can be used and taken on a daily basis as food and drink having an anti-new influenza virus action. The product of the present invention is effective in preventing infection with a new influenza virus and treating diseases caused by the new influenza virus.

It is a graph which shows the hemagglutination suppression effect of the karin extract CSD3. It is a graph which shows the A / Chiba / 1001/2009 (H1N1) pdm infectious neutralizing activity (plaque method) of the karin extract CSD3. It is a graph which shows the A / Chiba / 1001/2009 (H1N1) pdm infectious neutralizing activity (TCID ₅₀ method) of the karin extract CSD3.

  It is desirable to use the fruits of karin as the raw material for the product of the present invention.

  The method for obtaining the extract of the present invention from the pulverized plant is not particularly limited, but water, methanol, ethanol, n-propanol, and lower alcohols such as n-butanol, ether, ethyl acetate, acetone, glycerin, and propylene glycol. One or two or more mixed solvents of organic solvents such as these are added, and extraction is performed by a conventional extraction method. However, in consideration of taking the anti-influenza virus agent of the present invention orally, it is desirable to extract using water, ethanol or a mixture thereof from the viewpoint of safety.

  The extraction condition is not particularly limited, but is preferably about 1 to 5 hours at 50 to 90 ° C. The extract can be filtered and the extraction solvent can be distilled off, followed by concentration or lyophilization under reduced pressure. Further, those obtained by fractionating and purifying these extracts by organic solvent fractionation, column chromatography or the like can also be used.

  The form of use of the product of the present invention is not particularly limited, and powders, tablets, troches can be obtained by adding a solvent, a dispersant, a pharmaceutical carrier, an emulsifier, a diluent, a stabilizer, etc. to the plant extract exemplified as an active ingredient. Preparations, inhalants, mouthwashes, mouthwashes, suppositories, injections, etc., and can be prepared as an oral route, airway administration, intravenous administration, rectal administration, subcutaneous administration, skin Internal administration etc. can be illustrated. In this case, the dose to adults is preferably 10 to 2000 mg / day for each extract, but is not limited to this value. The amount of the extract added to various preparations varies depending on the form of the preparation, but it is preferable to add the extract so that the ratio is 0.001% by weight or more, preferably about 0.01% by weight or more.

  Moreover, the infection suppression article which can contribute to influenza prevention can be provided by adsorb | sucking, impregnating, and adding this invention to a mask, an air-conditioner filter, clothing, a wet tissue, a spray liquid, etc. The amount of adsorbed and added plant extract in these uses varies depending on the form of the infection-suppressing product, and cannot be generally defined, but is added so that the proportion is 0.001 to 5% by weight. Is preferred.

  Further, since the present invention is highly safe, for example, it is blended in confectionery such as chewing gum, candy, tablet confectionery, gummy jelly, chocolate and biscuits, ice cream, sorbet and other frozen confectionery, beverages, soups, jams and other foods and drinks, Can be used. The amount added varies depending on the form of use and the taste of the extract, but it is added in an amount of 0.001 to 5% by weight, preferably about 0.01 to 1% by weight, based on the food or drink. Is preferred.

  Hereinafter, the present invention will be specifically described with reference to examples and test examples, but the present invention is not limited thereto.

Example 1
Cell Line and Virus Preparation Cells were cultured in Eagle's minimum essential medium (EMEM) containing 10% fetal calf serum using Mardin-Darby canine kidney (MDCK) cells. As influenza viruses, A / Udorn / 307/72 (H3N2) and A / Chiba / 1001/2009 (H1N1) pdm were used. A / Udorn / 307/72 is inoculated into the chorioallantoic cavity of the growing chicken egg (11th day egg) and propagated, and Temperature-Sensitive Mutants of Influenza A / Udorn / 72 (H3N2) Virus. 1982). The A / Chiba / 1001/2009 (H1N1) pdm strain was inoculated into MDCK cells and grown for 2 days in a carbon dioxide incubator (37 ° C., 5% CO ₂ ). Centrifugation was performed at 5,000 rpm for 5 minutes, and the supernatant was used as a virus solution.

(Example 2)
Purification of active ingredients in Karin Dried fruits of Karin (C. sinensis Koehne) were obtained from the Hubei market in China. 100 g of dried fruit was refluxed and extracted with 50% ethanol (700 ml) for 1 hour. After filtration, the extract was concentrated under reduced pressure and freeze-dried to obtain a 50% ethanol extract of Cullin (CSE50) (23 g) . CSE50 is made turbid with water, and Diaion HP-20 (Mitsubishi Chemical) column chromatography (column: 8 × 15 cm) has 5 stages of hydrous ethanol (0%, 20%, 40%, 60%, 100% ethanol) CSD1 (water elution fraction: 14.5 g), CSD2 (20% ethanol elution fraction: 3.2 g), CSD3 (40% ethanol elution fraction: 3.7 g), CSD4 (60% ethanol elution fraction: 161 mg) and 5 fractions of CSD5 (100% ethanol elution fraction: 161 mg) were obtained. Condensed polyphenol in each fraction was quantified by vanillin-hydrochloric acid method using (-)-epicatechin as a standard.

^a；A/Udorn/307/72に対する50％感染中和濃度
^b；エピカテキン換算、n=3、平均値±標準偏差 (Example 3)
Measurement of polyphenol content and infection neutralization value of each karin fraction The infection neutralization value of each fraction was measured by plaque method using A / Udorn / 307/72 (H3N2). Add an equal volume of 10-fold serial dilution to 0.1 ml of virus solution (1,000 PFU / ml), react at room temperature for 30 minutes, inoculate 100 μl into MDCK cells (6-well plate), and incubate at room temperature for 60 minutes. Adsorbed for 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 culturing at 34 ° C. for 3 days, the number of plaques that appeared was counted, and the 50% infection neutralization concentration (IC ₅₀ ) was determined. 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 virus and fractions.
As in Example 2, each fraction of A / Udorn / 307/72 (H3N2) was obtained by eluting a 50% ethanol extract of karin with 5 stages of aqueous ethanol using DiaionHP-20 and fractionating it into CSD1-5. Table 1 shows the 50% infection neutralizing IC ₅₀ and the polyphenol content for each. The CSD3 fraction had the highest infection suppression effect (IC ₅₀ = 0.8 μg / ml). CSD3 had the highest polyphenol content of 53.8%.
Therefore, in subsequent experiments, CSD3 was used to evaluate the antiviral effect against the new influenza virus A / Chiba / 1001/2009 (H1N1) pdm.

^a ; 50% neutralizing concentration against A / Udorn / 307/72
^b : Equicatechin conversion, n = 3, average value ± standard deviation

Example 4
Hemagglutination reduction test against CSD3-treated new influenza virus A / Chiba / 1001/2009 (H1N1) pdm The hemagglutination titer (HA price) was measured using a 96-well plate. Make a serial dilution series (50 μl) of each concentration of CSD3-treated virus in phosphate buffered saline, add 50 μl of 0.5% (v / v) chicken erythrocytes, and allow to stand 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 value of each virus solution. The inhibitory effect of hemagglutination on the new influenza virus A / Chiba / 1001/2009 (H1N1) pdm in the Cullin CSD3 fraction was evaluated by the HA titration reduction test. Add 0.1 ml of virus solution (HA value: 32) to an equal volume of CSD3 solution (0: control, 0.1, 2, 10, 50, 500 μg / ml) and react at room temperature for 60 minutes. It was measured.
According to the above method, the effect of A / Chiba / 1001/2009 (H1N1) pdm on hemagglutination inhibition by CSD3 treatment was evaluated by a hemagglutination titration reduction test. The hemagglutination test is based on a reaction in which hemagglutinin on the surface of the virus binds to the receptor of erythrocytes, and is a test for evaluating the receptor binding ability of the virus. In CSD3 of 50 μg / ml or more, the hemagglutination effect of CSD3 itself was observed, so the hemagglutination titration reduction test for A / Chiba / 1001/2009 (H1N1) pdm was performed using CSD3 of 25 μg / ml or less. It was. At 1 μg / ml or less, the virus HA titer was 16 as in the untreated virus, and no reduction in hemagglutination titer was observed. In the treatment of 5 μg / ml, it was 8 and decreased to 1/2. At 25 μg / ml, it decreased below the detection limit (2) (Fig. 1). From the above results, the hemagglutination inhibitory effect by the new influenza of the karin fraction and the new influenza virus adsorption inhibitory effect on the cells were observed.

(Example 5)
Infectious titre reduction test of CSD3 against new influenza virus A / Chiba / 1001/2009 (H1N1) pdm Infectious titre reduction of infectious titer of kalin CSD3 fraction against new influenza virus A / Chiba / 1001/2009 (H1N1) pdm It was evaluated by testing. Add 0.1 ml of virus solution (2 × 10 ⁷ PFU / ml, HA value: 32) to an equal volume of CSD3 solution (0: control, 0.1, 2, 10, 50, 500 μg / ml) for 60 minutes at room temperature After the reaction, the infectious titer was measured by the plaque method and the Tissue Culture Infectious Dose 50% (TCID ₅₀ ) method.
i) Measurement by plaque method 10-fold serial dilutions of CSD3-treated virus at each concentration were prepared, 100 μl was inoculated into MDCK cells (6-well plate), and plaques were counted as described above. The infectious titer was expressed as PFU (plaque forming units) / ml.
ii) Measurement by TCID ₅₀ method Make 10-fold serial dilutions of CSD3-treated virus at various concentrations in MEM containing 2.5 μg / ml trypsin, inoculate 200 μl into MDCK cells (24-well plate), Incubated at 37 ° C for 3 days. After culturing, infection was determined by the presence or absence of hemagglutination activity in the supernatant, and the amount of TCID ₅₀ was determined.
In accordance with the method described above, the infection neutralizing activity was evaluated by an infectivity titer reduction test using the plaque method. The infectivity of untreated virus was 1.0 × 10 ⁷ PFU / ml. CSD3 treatment confirmed the infection-inhibiting effect in a dose-dependent manner, and the infection titer decreased to about 2/3 at 1 μg / ml, about 1/60 at 25 μg / ml, and about 1 / 10,000 at 250 μg / ml. (Figure 2). However, since there were many very small plaques and accurate measurement was difficult, it was evaluated by an infectious titration reduction test using the TCID ₅₀ method.
As a result of measurement by TCID ₅₀ method, about 1/3 with 1 μg / ml CSD3 treatment, about 1/10 with 5 μg / ml, about 1/100 with 25 μg / ml, 1 / 3,000 with 250 μg / ml Infectious titer was suppressed (Figure 2). When TCID ₅₀ was evaluated by observing cytopathic effect (CPE) under a microscope simultaneously with evaluation of TCID ₅₀ , the results were completely consistent.
With 5 μg / ml CSD3 treatment, the reduction in hemagglutination titer was about ½, but the reduction in infectivity titer was about 1/10. The decrease in infectious titer was 5 times the decrease in hemagglutinating titer, suggesting that CSD3 mainly suppresses the virus growth process after adsorption of the new influenza virus A / Chiba / 1001/2009 (H1N1) pdm.

  Based on the above results, the hemagglutination inhibitory activity and infectious neutralization activity against the new influenza virus A / Chiba / 1001/2009 (H1N1) pdm were evaluated using the active fraction CSD3 in Karin. It was revealed that the virus treated with CSD3 decreased the hemagglutination titer to about 1/2 and the infectivity to about 1/10. Treatment with 250 μg / ml reduced the infectious titer to 1 / 3,000. These results indicate that the anti-influenza virus active ingredient in karin is also effective against H1N1 novel influenza virus. Furthermore, since the infectivity decreased more than the decrease in the hemagglutination titer, the existence of an inhibitory action after the virus adsorption stage was suggested.

  Using the Karin extract prepared in Example 1, mouthwash, inhalant, troche, spray, chewing gum, candy, tablet confectionery, beverage, powder, tablet, gargle, gummy jelly, chocolate, biscuit, ice , Sherbet, soup, jam, wet tissue and mask were prepared. The prescription was shown as an Example below.

(Example 6)
Prescription ethanol for mouthwash 2.0% by weight
Fragrance 1.0% by weight
Saccharin 0.05 wt%
Chlorhexidine hydrochloride 0.01% by weight
CSD3 0.5 wt%
Water remaining
100.0% by weight

(Example 7)
Prescription ethanol for inhalation 5.0 wt%
CSD3 1.0 wt%
Water remaining
100.0% by weight

(Example 8)
Lozenge prescription glucose 72.3% by weight
Lactose 19.0% by weight
Gum arabic 6.0% by weight
Fragrance 1.0% by weight
Sodium monofluorophosphate 0.7% by weight
CSD3 1.0 wt%
100.0% by weight

Example 9
Prescription ethanol of spray liquid 25.0% by weight
Citric acid 1.5% by weight
Trisodium citrate 1.0 wt%
CSD3 0.5 wt%
Water remaining
100.0% by weight

(Example 10)
Chewing gum formula gum base 20.0% by weight
54.7% by weight sugar
Glucose 15.0% by weight
Minamata 9.3 wt%
Fragrance 0.5% by weight
CSD3 0.2 wt%
100.0% by weight

(Example 11)
Candy prescription sugar 50.0% by weight
Minamata 34.0 wt%
Citric acid 1.0% by weight
Fragrance 0.2 wt%
CSD3 0.4 wt%
Water remaining
100.0% by weight

(Example 12)
Prescription sugar for tablet candy 76.1% by weight
Glucose 19.0% by weight
Sucrose fatty acid ester 0.2% by weight
Fragrance 0.2 wt%
CSD3 0.5 wt%
Water remaining
100.0% by weight

(Example 13)
Beverage prescription orange juice 30.00% by weight
Isomerized sugar 15.24% by weight
Citric acid 0.10% by weight
Vitamin C 0.04% by weight
Perfume 0.10% by weight
CSD3 0.10 wt%
Water remaining
100.00% by weight

(Example 14)
Formulation of powdered corn starch 55.0% by weight
Carboxycellulose 40.0% by weight
CSD3 5.0 wt%
100.0% by weight

(Example 15)
Tablet prescription lactose 70.0% by weight
Crystalline cellulose 15.0% by weight
Magnesium stearate 5.0 wt%
CSD3 10.0 wt%
100.0% by weight

(Example 16)
Prescription of gargle ethanol 2.00% by weight
Perfume 1.00% by weight
Saccharin 0.05 wt%
Chlorhexidine hydrochloride 0.01% by weight
CSD3 0.50 wt%
Water remaining
100.00% by weight

(Example 17)
Gummy jelly prescription gelatin 60.00 wt%
Minamata 23.00 wt%
Sugar 8.50% by weight
Vegetable oil and fat 4.50% by weight
Mannitol 2.95% by weight
Lemon juice 1.00% by weight
CSD3 0.05 wt%
100.00% by weight

  The present invention can be widely used in daily life as a new influenza virus infection suppression product by adsorbing, impregnating and adding to a mask, air conditioner filter, clothing, wet tissue, spray solution, and the like. Furthermore, it can be added to various foods and drinks, and can be used and taken on a daily basis as foods and drinks having an anti-new influenza virus action. The product of the present invention is effective in preventing infection with a new influenza virus and treating diseases caused by the new influenza virus. In other words, it can be expected to be applied as a new product development material as an infection prevention material.

Claims (5)

  An anti-influenza virus agent characterized by containing an extract of karin (Chaenomeles sinensis, Pseudocydonia sinensis) as an active ingredient.   An anti-new influenza virus agent characterized in that an extract obtained by extracting karin with 50% ethanol water and purifying the extract by column fractionation is used as an active ingredient.   An inhibitor of erythrocyte aggregation caused by a novel influenza virus, characterized in that an extract obtained by extracting karin with 50% ethanol water and purifying the extract by column fractionation is used as an active ingredient.   An inhibitor of adsorption of new influenza virus to cells, characterized in that an extract obtained by extracting kalin with 50% ethanol water and purifying the extract by column fractionation is used as an active ingredient. .   A food and drink having an anti-new influenza virus action containing a karin extract.

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