TW201304797A - Pandemic influenza antiviral agent - Google Patents

Abstract

A pandemic influenza antiviral agent, containing as an active ingredient an extract obtained by extracting Chinese quince using 50% ethanol-water and purifying the extract solution by column fractionation.

Description

Anti-new influenza virus agent

The present invention relates to an anti-new influenza virus agent which is an active ingredient of a plant-derived extract having a novel influenza virus infection inhibitor, a novel influenza virus red blood cell agglutination inhibitor, and a novel influenza virus pair. Adsorption inhibitor of cells.

In the case of the influenza virus which is the epidemic of the influenza, it is an RNA virus having a membrane of about 1 million in diameter. The different types of antigenicity are classified into types A, B, and C, and the types A and B of the epidemic spread can be seen. On the surface of these virions, two kinds of glycoproteins, which are red blood cell agglutinin (HA) and neuraminidase (NA), are rhomboidate, and there are eight gene RNAs in the interior. HA and NA on the surface of the virus frequently cause mutations in the same subtype, and new antigenic variants appear almost every year.

The influenza virus ejected from the cough droplets invades from the nose or mouth of the human body, and is adsorbed to the mucosal epithelial cells of the upper respiratory tract by the thorny glycoprotein HA of the surface of the virus, and begins to proliferate after the cells invade. Through recent research, the mechanism of infection of the virus has become clear. The virus system binds to a receptor composed of a sugar chain present in the surface layer of a human target cell, and is ingested into an endosome, and is invaded into the cell by fusion of the viral membrane and the endosome membrane, and is subjected to shelling/transfer. The expression and replication of the viral gene occurs, and finally, by proliferating from the host cell membrane to form progeny virions.

Infected by the influenza virus, symptoms such as sudden fever, headache, joint pain, and generalized burnout may occur in a few days. Respiratory symptoms such as cough or sore throat, nasal water, and stuffy nose may occur before and after. Unlike the so-called cold, it is characterized by strong infection and can cause an explosive epidemic in a short period of time. Moreover, the structure of the HA protein of the influenza virus is repeated every year, and the antibodies produced by the past infections do not have much effect, which is also the cause of the spread of the infection.

In order to suppress the infection of influenza virus, it is considered that the inhibition of the adsorption of epithelial cells, the inhibition of invasion of cells, the inhibition of gene transcription/replication, the inhibition of protein synthesis, the inhibition by cell release, etc., respectively become antiviral drugs. Target. So far, antiviral drugs such as amantadine, rimantadine, and zanamivir have been developed, but allergies, neuropsychiatric symptoms, gastrointestinal symptoms, and autonomic nervous systems have been reported. Side effects such as symptoms, need to pay attention to its application.

Moreover, it is difficult to suppress infection by vaccination from the infection and proliferation of influenza virus epithelium in the respiratory mucosa, or the fact that the epidemic type of the year cannot be correctly predicted. At present, the most effective prevention strategies are considered to be frequent gargles, to avoid dry throat, adequate nutrition and rest. The anti-influenza virus agent that can be utilized in daily life is expected to have a high infection inhibitory effect and no safety problem.

In recent years, as an anti-influenza material derived from natural products, polyphenols of tea or black tea have been reported (Non-Patent Documents 1 and 2), and it has been clearly known by the use of human trials that the mouthwash of black tea actually inhibits viral infection. Non-special Li literature 3). In addition, the flavonoid component derived from Astragalus membranaceus has been reported to exhibit an influenza infection suppressing effect by the sialidase inhibitory activity of the virus (Non-Patent Document 4). Further, a medicinal preparation of Guizhi Eryue Yuyi Tang (Patent Document 1), black currant extract (Patent Document 2), potato anthocyanin pigment (Patent Document 3), and guava leaf extract (Patent Document 4) The antiviral effects of Apocynum venetum L. extract (Patent Document 5) and olive leaf extract (Patent Document 7) have also been reported.

In addition, in the Rosaceae plant, an anti-influenza agent containing an extract of flower buds or petals as an active ingredient is also disclosed (Patent Document 6), and an extract of papaya papaya (Patent Document 8), light papaya is reported. The effect of the anti-influenza virus effect of the column-restricted substance (Patent Document 9 and Non-Patent Document 5) and the infection effect of the light-skin papaya substance of the plaque test method on the novel influenza virus (Non-Patent Document 6) Further, the papaya extract inhibits aggregation of red blood cells caused by the influenza virus (Non-Patent Document 7).

Chaenomeles sinensis is a deciduous tree native to China. The fruit is made into light papaya or sugar stains, syrup and so on. This fruit is called papaya fruit and is prescribed as a traditional Chinese medicine for removing phlegm, antitussive, and analgesic. Identification of the medicinal ingredients in the fruit of Papaya, and the report that there are triterpenoids such as oleanolic acid as antibacterial components of Streptococcus pyogenes against pharyngitis. Polyphenols act as anti-inflammatory ingredients. The inventors of the present invention reported the erythrocyte aggregation inhibiting effect and the infection suppressing effect of the high molecular polyphenols in the light papaya fruit on the seasonal influenza virus (Patent Document 9).

However, there is no report on the effect of the column division of the light papaya on the red blood cell agglutination caused by the novel influenza virus and the adsorption inhibition effect of the novel influenza virus on the cells, as shown in the invention patent. It was first explained by the present invention. Influenza has become popular almost every year, sometimes causing a worldwide pandemic called a pandemic. In March 2009, influenza caused by the new H1N1 virus that started in Mexico was also one of them. This time, the red blood cell agglutination inhibitory effect and infection inhibitory effect of the novel influenza virus A/Chiba/1001/2009 (H1N1) pdm were observed in the papaya extract.

[Previous Technical Literature] [Patent Literature]

[Patent Document 1] Japanese Patent Laid-Open No. Hei 6-196980

[Patent Document 2] Japanese Patent Laid-Open Publication No. 2000-212092

[Patent Document 3] Japanese Patent Laid-Open Publication No. 2001-316399

[Patent Document 4] Japanese Patent Laid-Open Publication No. 2000-273048

[Patent Document 5] Japanese Patent Laid-Open No. Hei 11-71296

[Patent Document 6] Japanese Patent Laid-Open Publication No. 2002-145790

[Patent Document 7] Japanese Special Table 2002-020305 Bulletin

[Patent Document 8] Japanese Patent Laid-Open Publication No. 2005-343836

[Patent Document 9] PCT/JP2010/005762 Specification

[Non-patent literature]

[Non-Patent Document 1] Journal of Infectious Diseases, 68 (7) 824-829 (1994)

[Non-Patent Document 2] Journal of Infectious Diseases, 70 (11) 1190-1192 (1996)

[Non-Patent Document 3] Journal of Infectious Diseases, 71 (6) 487-494 (1997)

[Non-Patent Document 4] Chem. Pharm. Bull. 38 (5) 1329-1332 (1990)

[Non-Patent Document 5] Journal of Ethnopharmacology, 118, 108-112 (2008)

[Non-Patent Document 6] Foodnews, 43(1) 155-157 (2009)

[Non-Patent Document 7] J Agric. Food Chem. 53, 928-934

The object of the present invention is to provide a safe and high-efficiency plant extract which can be safely used daily, to provide a novel influenza virus infection inhibitor, a novel influenza virus-inducing inhibitor of red blood cell agglutination, and a novel influenza virus adsorption to cells. Inhibitor.

In order to solve the above problems, the inventors of the present invention have focused on papaya papaya which is a side effect and has high safety, and has found a light papaya extract in MDCK cells to which a novel influenza virus is added, that is, in a light papaya fruit. Polyphenol-rich fraction (CSD3), infective neutralizing activity against the new influenza virus A/Chiba/1001/2009 (H1N1) pdm; and the discovery of a part of the genus Papaya (CSD3) The red blood cell agglutination inhibitory effect on the novel influenza virus is completed, and the present invention is completed.

That is, the present invention is a novel influenza virus infection inhibition Agent, a red blood cell agglutination inhibitor in a novel influenza virus-infected cell, and an adsorption inhibitor of a novel influenza virus to a cell, characterized in that an extract of Chaenomeles sinensis (Pseudocydonia sinensis) is used as an active ingredient. Further, the present invention is a food or drink having an action against a novel influenza virus, which comprises a light papaya extract.

The present invention provides a highly safe light papaya extract as an active ingredient, and provides a strong influenza virus infection inhibitor against a novel influenza virus, a red blood cell agglutination inhibitor in influenza virus-infected cells, and a novel type. Inhibitors of the adsorption of influenza virus to cells. Moreover, the papaya extract of the active ingredient of the present invention has high safety, so it can be infected as a novel influenza virus by adsorption, impregnation, addition to a mask, cold air filter, clothing, wet tissue, spray, and the like. It is widely used in daily life by suppressing products. Further, it can be added to foods and drinks such as chewing gum, candy, ingot, and beverage, and can be used and taken daily as a food or drink having a function against a novel influenza virus. The strain of the present invention is effective for the prevention of infection of a novel influenza virus or the treatment of a disease caused by a novel influenza virus.

In the papaya of the raw material of the present invention, it is preferred to use the fruit.

The method for obtaining the extract of the present invention from the pulverized material of the above plant Specially defined as a mixed solvent of water, methanol, ethanol, n-propanol and n-butanol; ether; ethyl acetate; acetone; glycerin; propylene glycol and other organic solvents, or a mixture of two or more Know the extraction method to extract. However, in view of the oral ingestion of the anti-influenza virus agent of the present invention, it is preferred to use ethanol or a mixture thereof for extraction in terms of safety.

Although the extraction conditions are not particularly limited, it is preferably from 50 to 90 ° C for about 1 to 5 hours. The extract is filtered, and after extracting the extraction solvent, it can be used for concentration or freeze-drying under reduced pressure. Further, it is also possible to use those extracts which have been distinguished by an organic solvent, a chromatography method, or the like.

The form of use of the product of the present invention is not particularly limited, and may be prepared as a powder or an ingot by adding a solvent, a dispersing agent, a carrier for a formulation, an emulsifier, a diluent, a stabilizer, etc. to an exemplary plant extract as an active ingredient. Agent, tablet (troche), inhalant, mouthwash, gargle, suppository, injection, etc., the administration route can be exemplified by oral administration, respiratory administration, intravenous administration, rectal administration, subcutaneous Investment, intradermal administration, etc. At this time, the dose to the adult is 10 to 2000 mg/day for each extract, but is not limited to this value. The amount of the extract added to the various preparations of the extract varies depending on the form of the preparation, but is preferably added in a ratio of 0.001% by weight or more, preferably about 0.01% by weight or more.

Further, by adsorbing, impregnating, and adding the present invention to a mask, a cold air filter, clothes, a wet tissue, a liquid spray, or the like, it is possible to provide an infection suppressing product which can contribute to the prevention of influenza. The amount of adsorption and addition of the plant extract in such applications varies depending on the form of the infection-inhibiting article, and The law is generally prescribed, but it is suitable to be added in a ratio of 0.001 to 5% by weight.

Moreover, since the present invention has high safety, it can be blended with desserts such as chewing gum, candy, ingot, gummy candy, chocolate, biscuits, etc.; cold desserts such as ice cream and slush; and foods such as beverages, soups, jams, etc. Used in. Although the amount to be added differs depending on the form of use and the taste property of the extract, it is preferably added in a ratio of 0.001 to 5% by weight, preferably about 0.01 to 1% by weight, based on the food and drink.

[Examples]

The present invention will be more specifically described below by way of examples and test examples, but the invention is not limited thereto.

[Example 1] Modulation of cell lines and viruses

The cell line was cultured using Mardin-Darby canine kidney (MDCK) cells in Eagle's minimum essential medium (EMEM) containing 10% fetal calf serum. The influenza virus system uses A/Udorn/307/72 (H3N2) and A/Chiba/1001/2009 (H1N1) pdm. A/Udorn/307/72 is inoculated into the chorioallantoic cavity of the developing egg (11th egg) to proliferate, and by Temperature-Sensitive Mutants of Influenza A/Udorn/72(H3N2)Virus.Virology 117,45- Refined by the method described in 61 (1982). The A/Chiba/1001/2009 (H1N1) pdm strain was inoculated into MDCK cells and allowed to proliferate for 2 days in a carbon dioxide gas culture machine (37 ° C, 5% CO ₂ ). The cells were centrifuged at 5,000 rpm for 5 minutes, and the supernatant was used as a virus solution.

[Embodiment 2] Refining of active ingredients in light papaya

The dried fruit of C. sinensis Koehne is obtained from the market in Hubei Province, China. 100 g of dried fruit was reflux-extracted with 50% ethanol (700 ml) for 1 hour, and after filtration, the extract was concentrated under reduced pressure and lyophilized to obtain a 50% ethanol extract (CSE50) (23 g) of papaya. CSE50 was turbid by water, using Diaion HP-20 (Mitsubishi Chemical) chromatography (column: 8 x 15 cm) with 5 stages of aqueous ethanol (0%, 20%, 40%, 60%, 100% ethanol) Dissolution, CSD1 (water-soluble 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), CSD5 were obtained. 5 parts (100% ethanol elution fraction: 161 mg). The condensed polyphenol in each fraction was quantified by the vanillin-hydrochloric acid method using (-)-epicatechin as a standard.

[Example 3] Determination of polyphenol content and infection neutralization price of each light papaya

The infection neutralization price of each fraction was determined by the speckle method using A/Udorn/307/72 (H3N2). Add 10 parts of the same fraction of the 10 ml stage dilution to 0.1 ml of virus solution (1,000 PFU/ml), react at room temperature for 30 minutes, and inoculate 100 μl of the cells into MDCK cells (6-well plate) for adsorption at room temperature. 60 minutes. After the 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 to be solidified. After culturing at 34 ° C for 3 days, the number of spots formed was measured, and a 50% infection neutralization concentration (IC ₅₀ ) was determined. The dilution of the virus and the fraction was 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).

As in Example 2, the 50% ethanol extract of Papaya was dissolved in 5 stages of aqueous ethanol using Diaion HP-20, and was divided into 50 parts of CSD1~5 to A/Udorn/307/72 (H3N2). The % of infection and the IC ₅₀ and polyphenol content are shown in Table 1. The CSD3 fraction had the highest infection inhibitory effect (IC ₅₀ = 0.8 μg/ml). In addition, the polyphenol content of CSD3 was 53.8%, which was the highest.

Therefore, in the subsequent experiments, CSD3 was used to evaluate the antiviral effect against the novel influenza virus A/Chiba/1001/2009 (H1N1) pdm.

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

b; converted with epicatechin, n=3, mean±standard deviation

[Example 4] Red blood cell agglutination reduction test for CSD3 treated influenza virus A/Chiba/1001/2009(H1N1)pdm

The measurement of the red blood cell agglutination power (HA price) was carried out using a 96-well plate. A 2-fold dilution series (50 μl) of CSD3-treated virus at various concentrations was prepared in phosphate buffered saline, 50 μl of 0.5% (v/v) chicken red blood cells was added, and after standing at 4 ° C for 1 hour, the presence or absence of red blood cell agglutination was determined. The reciprocal of the highest dilution showing agglutination was used as the HA value of each virus solution. The erythrocyte agglutination inhibitory effect of the new influenza virus A/Chiba/1001/2009 (H1N1) pdm was evaluated by the HA valence reduction test. An equivalent amount of CSD3 solution (0: control group, 0.1, 2, 10, 50, 500 μg/ml) was added to 0.1 ml of the virus solution (HA price: 32), and the mixture was allowed to react at room temperature for 60 minutes, and then the HA value was measured.

According to the above method, the red blood cell agglutination cost reduction test is used to evaluate Red blood cell agglutination inhibitory effect of A/Chiba/1001/2009 (H1N1) pdm treated with CSD3. The red blood cell agglutination test is a test based on the binding of hemagglutinin on the surface of a virus to a receptor of a red blood cell, and is an assay for evaluating the receptor binding ability of a virus. In CSD3 of 50 μg/ml or more, erythrocyte agglutination of CSD3 itself was observed. Therefore, the red blood cell agglutination valence reduction test of A/Chiba/1001/2009 (H1N1) pdm was carried out using CSD3 of 25 μg/ml or less. The HA value of the virus at the same time as 1 μg/ml was 16 as compared with the untreated virus, and no decrease in the red blood cell agglutination price was observed. At 5 μg/ml, the treatment was 8 and the decrease was 1/2. At 25 μg/ml, it was reduced to the detection limit (2) (Fig. 1). From the above results, it was observed that the inhibition of the red blood cell agglutination caused by the novel influenza by the papaya fraction and the adsorption inhibition effect of the novel influenza virus on the cells were observed.

[Example 5] Infectivity test for CSD3 of the new influenza virus A/Chiba/1001/2009(H1N1)pdm

The infectious neutralization effect of the CSD3 part of the light papaya on the novel influenza A/Chiba/1001/2009 (H1N1) pdm was evaluated by the infectivity reduction test. Add an equal amount of CSD3 solution (0: control group, 0.1, 2, 10, 50, 500 μg/ml) to 0.1 ml of virus solution (2×10 ⁷ PFU/ml, HA price: 32) and react at room temperature. Infected price was determined by the oil plaque method and the Tissue Culture Infectious Dose 50% (TCID ₅₀ ) method for 60 minutes.

i) Determination of the plaque method

A 10-fold serial dilution series of CSD3-treated virus at each concentration was prepared, and 100 μl was inoculated into MDCK cells (6-well plate), and the plaques were counted by the aforementioned method. Infectivity is expressed in terms of PFU (plaque forming units)/ml.

Ii) Determination of TCID ₅₀ method

A 10-fold serial dilution series of CSD3-treated virus at each concentration was prepared in MEM containing 2.5 μg/ml trypsin, and 200 μl was seeded in MDCK cells (24-well plate), and cultured in a carbon dioxide gas culture machine at 37 ° C for 3 days. After the culture, the infection was determined by the presence or absence of the red blood cell agglutination activity in the supernatant, and the amount of TCID _{50 was} determined.

Infectious neutralizing activity was evaluated by the plaque method of the infectivity valence test in accordance with the above method. The infectious power of the untreated virus was 1.0 × 10 ⁷ PFU/ml. By CSD3 treatment, the inhibitory effect of the dose was confirmed. When the infectious power was reduced to about 2/3 and 25 μg/ml at 1 μg/ml, the infectivity was reduced to about 1/60, 250 μg/ml. The infectious power price was reduced to approximately 1/10,000 (Figure 2). However, there are many very small spots that are difficult to measure correctly, and therefore are evaluated by the TCID ₅₀ method of the infectious power price reduction test.

As a result of the measurement by the TCID ₅₀ method, when the infectious power of 1 μg/ml of CSD3 was inhibited to about 1/3 and 5 μg/ml, the infectivity was suppressed to about 1/10 and 25 μg/ml, and the infectious power was suppressed to The infectivity price was suppressed to 1/3,000 at about 1/100 and 250 μg/ml (Fig. 2). Further, within cytopathic effect under the microscope and the TCID ₅₀ Evaluation simultaneously (Cytopathic effect: CPE) was observed after the TCID _50, of the results being entirely consistent.

At 5 μg/ml of CSD3, the decrease in red blood cell agglutination power was about 1/2, but the decrease in infectious power was about 1/10. The decrease in the infectivity price is five times the decrease in the red blood cell agglutination price, suggesting that CSD3 mainly inhibits the virus proliferation process after adsorption of the novel influenza virus A/Chiba/1001/2009 (H1N1) pdm.

From the above results, after the erythrocyte agglutination inhibitory activity and the infectious neutralization activity of the novel influenza virus A/Chiba/1001/2009(H1N1)pdm were evaluated using the active fraction CSD3 in papaya papaya, it was clearly found that 5 μg was obtained. /ml of CSD3 treated virus, the red blood cell agglutination power price was reduced to about 1/2, and the infectivity was reduced to about 1/10. When treated at 250 μg/ml, the infectious power price was reduced to 1/3,000. These results show that the anti-influenza virus active ingredient in Papaya is also effective against the H1N1 novel influenza virus. Further, the decrease in the infectivity is greater than the decrease in the erythrocyte agglutination valence, suggesting that the inhibitory effect after the virus adsorption phase exists.

Using the papaya extract prepared in Example 1, preparing a mouthwash, an inhaler, a tablet, a spray, a chewing gum, a candy, an ingot, a drink, a powder, a lozenge, a tincture, a gummy, a chocolate , biscuits, ice, slush, soup, jam, wet wipes, masks. The formulation is shown below as an example.

[Embodiment 6] 漱口药处方

[Embodiment 7] Inhalant

[Embodiment 8] Tablet place

[Embodiment 9] Spraying place

[Embodiment 10] Chewing gum place

[Example 11] Candy place

[Embodiment 12] Ingot sugar

[Example 13] Drink place

100.00% by weight

[Embodiment 14] Powder place

[Example 15] Lozenge

[Example 16] Ingredient

[Example 17] Gummy candy square

[Industrial availability]

The present invention can be widely used in daily life as a novel influenza virus infection suppressing product by adsorption, impregnation, addition to a mask, cold air filter, clothes, wet tissue, spray, and the like. Further, it can be added to various foods and drinks, and can be used and ingested daily as a food and drink having a function against a novel influenza virus. The strain of the present invention is effective for the prevention of infection of a novel influenza virus or the treatment of a disease caused by a novel influenza virus. In other words, it is expected to be used as a material for new product development as an infection prevention material.

Fig. 1 is a graph showing the erythrocyte agglutination inhibitory effect of the light papaya extract CSD3.

Fig. 2 is a graph showing the A/Chiba/1001/2009 (H1N1) pdm infectious neutralization activity (spotting method) of the light papaya extract CSD3.

Fig. 3 is a graph showing the A/Chiba/1001/2009 (H1N1) pdm infectious neutralization activity (TCID ₅₀ method) of the light papaya extract CSD3.

Claims (5)

An anti-new influenza virus agent characterized by the extract of Chaenomeles sinensis (Pseudocydonia sinensis) as an active ingredient. An anti-new influenza virus agent characterized by extracting light papaya using 50% ethanol water, and extracting the extract by using the extract to obtain an extract as an active ingredient. An inhibitor of red blood cell agglutination caused by a novel influenza virus characterized by extracting papaya using 50% ethanol water, and extracting the extract by using the extract to obtain an extract as an active ingredient . An inhibitor of the adsorption of cells by a novel influenza virus, characterized in that the papaya is extracted with 50% ethanol water, and the extract obtained by the column separation is refined to obtain an extract as an active ingredient. A food or drink having the function of resisting a novel influenza virus, which comprises a light papaya extract.