KR20120087928A - Anti-influenza virus agent - Google Patents

Abstract

Inhibitors of influenza virus adsorption, virus mRNA synthesis inhibitors in influenza virus infected cells, virus vRNA synthesis inhibitors in influenza virus infected cells, and viral cRNAs in influenza virus infected cells, using safe plant extracts that can be used with confidence in daily use Provision of Synthetic Inhibitors, Hemolytic Inhibitors in Viral Membrane Fusion Activity Tests, and Envelope Destructors of Influenza Viruses. An anti-influenza virus agent characterized by extracting the Chinese quince using 50% ethanol water and extracting the extract by column fractionation and purifying the extract.

Description

Anti-influenza virus agent {ANTI-INFLUENZA VIRUS AGENT}

The present invention provides an anti-influenza virus agent, an influenza virus adsorption inhibitor, an inhibitor of virus mRNA synthesis in influenza virus infected cells, and a virus vRNA synthesis inhibitor in influenza virus infected cells, comprising as an active ingredient extracts derived from plants having an infection inhibitory effect of influenza virus. , Inhibitors of viral cRNA synthesis in influenza virus infected cells, hemolytic inhibitors in viral membrane fusion activity tests, and envelope destroyers of influenza viruses.

The influenza virus that causes the pandemic of influenza every year is an RNA virus having an envelope membrane about 10,000 millimeters in diameter. The antigenic differences are classified into three types, A, B, and C, but the most prevalent spreads are A and B. 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 8 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 as each year.

The influenza virus released by the cough droplets invades the human nose or mouth, adsorbs to the mucosal epidermal cells of the upper airway by the spiked glycoprotein HA of the viral surface layer, and then invades the cells. Initiate proliferation. Recent studies have revealed the infection mechanism of viruses. The virus binds to receptors consisting of sugar chains present on the surface of human target cells, is housed in endosomes, invades the cells by fusion of the viral membrane and the endosomal membrane, and through decontamination and execution, the expression of viral genes and 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 systemic malaise within a few days, and before and after it, respiratory symptoms such as cough, sore throat, runny nose, and stuffy nose appear. It is different from the so-called cold, and its infectivity is strong, and it is characterized by an explosive fashion in a short time. In addition, the structure of the HA protein of influenza virus is mutated every year, and the fact that antibodies produced by past infections do not help much has become a factor in spreading the infection.

In order to suppress the infection of influenza virus, inhibition of the adsorption to epidermal cells, inhibition of invasion to cells, inhibition of gene transcription and replication, inhibition of protein synthesis, inhibition of release from cells are considered. It is targeted for viral medicine. To date, antiviral drugs such as amantazin, rimantazin, and zanamiville have been developed, but side effects such as hypersensitivity, mental neurological symptoms, digestive system symptoms, and autonomic nervous system symptoms have been reported. need.

Influenza viruses are also thought to be difficult to suppress infection by inoculation of vaccines, since the influenza virus infects and proliferates in the airway mucosal epidermis, and the epidemic of the year cannot be accurately predicted. Frequent oral cleaning, avoiding dry throat, getting plenty of nutrition and rest are currently considered the most effective precautions. 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 oral washing of black tea suppresses the actual viral infection by a human test. (Non-Patent Document 3). In addition, it is reported that the flavonoid component derived from gold exhibits 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), and guava leaf extract, which are herbal preparations ( Antiviral effects, such as patent document 4) and a napoma extract (patent document 5), are reported.

Moreover, in the rosacea plant, 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 the quince (patent document 8), and the column fraction of the quince (nonpatent literature 5). Influenza virus effects have been reported. However, the effect of inhibiting influenza virus adsorption of the column fractions of the quince in the present invention patent, the effect of inhibiting viral mRNA synthesis in influenza virus infected cells, the effect of inhibiting viral vRNA synthesis in influenza virus infected cells, the viral cRNA in influenza virus infected cells The report on the effect of inhibiting the synthesis, the effect of inhibiting hemolysis in the virus membrane fusion activity test, and the effect of envelope destruction of the influenza virus was not seen, and was first discovered by the present invention.

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

 Infectious Disease Magazine, 68 (7) 824-829 (1994)  Infectious Disease Magazine, 70 (11) 1190-1119 (1996)  Infectious Disease Magazine, 71 (6) 487-494 (1997)  Chem.Pharm.Bull. 38 (5) 1329-1332 (1990)  Journal of Ethnopharmacology, 118, 108-112 (2008)

An object of the present invention is to influenza virus adsorption inhibitors, virus mRNA synthesis inhibitors in influenza virus infected cells, virus vRNA synthesis inhibitors in influenza virus infected cells, influenza virus using high-safety plant extracts that can be used with confidence in daily life To provide viral replication inhibitors in infected cells, hemolytic inhibitors in viral membrane fusion activity tests and envelope disruptors of influenza viruses.

In order to solve the above problems, the present inventors focus on quince without high side effects and high safety, and analyze the RT-PCR method of mRNA, vRNA, cRNA in MDCK cells added with influenza virus, hemolytic reaction using chicken erythrocytes, virus The effect of the quince extract on the electron microscopic observation of the particles was found to complete the present invention.

That is, the present invention is an influenza virus adsorption inhibitor, a virus mRNA synthesis inhibitor in influenza virus infected cells, a virus vRNA synthesis inhibitor in influenza virus infected cells, characterized in that the extract of the Chinese quince (Chaenomeles sinensis, Pseudocydonia sinensis) is an active ingredient. , Inhibitors of viral cRNA synthesis in influenza virus infected cells, hemolytic inhibitors in viral membrane fusion activity tests, and envelope destroyers of influenza viruses. In addition, the present invention is a food and drink having an anti-influenza virus action containing a Chinese quince extract.

The present invention uses an extract of a high-fidelity quince as an active ingredient, an influenza virus adsorption inhibitor that is strong against influenza viruses, a virus mRNA synthesis inhibitor in influenza virus infected cells, a virus vRNA synthesis inhibitor in influenza virus infected cells, and an influenza virus infection To provide a virus replication inhibitor in a cell, a hemolytic inhibitor in a viral membrane fusion activity test and an envelope destroyer of influenza virus. Moreover, since the extract of the Chinese quince which is an active ingredient of the present invention has high safety, it can be widely used in daily life as an influenza virus infection suppressing product by adsorbing, impregnating and adding to masks, air conditioner filters, clothes, wet tissues, and spray liquids. . In addition, it can be added to foods such as chewing gum, candy, tablets, and beverages, and can be used and consumed daily as foods having an anti-influenza virus action. The present invention is effective for preventing infection of influenza virus and for treating diseases caused by influenza virus.

Figure 1 shows the disruptive activity of the quince extract against the viral envelope membrane.
2 shows adsorption inhibitory activity, vRNA synthesis inhibitory activity, mRNA synthesis inhibitory activity, cRNA synthesis inhibitory activity of Chinese quince extract.
3 shows the hemolytic inhibitory activity of Chinese quince extract.

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 pulverized 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 from the viewpoint 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, the extractant is distilled off, and then concentrated or lyophilized under reduced pressure can be used. Moreover, what refine | purified 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, and mouthwashes are 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 can be prepared as any preparation, such as a water-containing, suppository, injection, etc., 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 10-2000 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 at a ratio of at least 0.001% by weight, preferably at least about 0.01% by weight.

In addition, by adsorbing, impregnating, and adding the present invention to a mask, air conditioner filter, clothing, wet tissue, and spray solution, an infection suppressing article that can contribute to influenza prevention can be provided. The amount of adsorption and addition of the plant extract in these applications differs depending on the form of the infection suppressing article, and cannot be defined uniformly, but it is preferably added in a ratio of 0.001-5% by weight.

In addition, since the present invention has high safety, for example, chewing gum, candy, sweets, gummi jelly, chocolate, biscuits, confectionery, ice cream, sherbet, ice cream, beverages, soups, jams, and the like are commonly used. It is possible. As addition amount, although it changes with the usage form and the taste of the extract, it is preferable to add so that it may become 0.001-5 weight% with respect to food-drinks, Preferably it is about 0.01-1 weight%.

Although an Example and a test example are given to the following and this invention is concretely demonstrated to it, this invention is not limited to these.

Example 1

(Preparation of Chinese quince extract)

The dry pulverized product of Chinese quince was extracted with 50% EtOH, and then fractionated using Diion HP-20, and five fractions (CSD1-5) were obtained. Among these fractions, CSD3 (40% EtOH eluted fraction) having the highest activity was used as a sample.

That is, the reflux condenser was attached to the flask to which 300 g of quince fruit dried products and 3000 ml of 50% ethanol were attached, and it extracts, refluxing for 1 hour. The obtained extract was separated by filtration to remove the solvent, and then freeze-dried to obtain 69 g of the extract.

The quince 50% ethanol extract was provided on a Diaion HP20 (manufactured by Mitsubishi Chemical) column. It was eluted in the order of water, 20% ethanol, 40% ethanol, 60% ethanol. The component (CSD3) eluted with this 40% ethanol aqueous solution was extract | collected. The yield of this fraction was about 8.6 g as dry weight. The phenol content by the vanillin hydrochloric acid method of this product was 54% [(-)-epicatechin as a standard], and it was confirmed that it mainly contains a phenolic substance.

Example 2

Virus adsorption inhibitory effect, vRNA synthesis inhibitory effect, mRNA synthesis inhibitory effect, and cRNA synthesis inhibitory effect by CSD3 were extracted and analyzed by RT-PCR method. What dissolved CSD3 in 50% ethanol so that it might become 50 mg / mL was used as the sample stock solution. Dilute the sample stock with Tris-Glucose-Saline (TGS) (up to 10 ² to 10 ^6- fold dilution), 50 μl of these sample dilutions and virus solution (4 × 10 ⁴ to 4 × 10 ⁶ PFU (plaque forming unit) 50 µl of the mixture was reacted at room temperature for 10 minutes. 0.1 ml of this was inoculated into a monolayer culture (6-hole plate) of Madin-Darby canine kidney (MDCK) cells to which 0.4 ml of TGS was added. The virus was adsorbed at room temperature for 30 minutes, and the supernatant was removed after the reaction. Culture medium was added, and after culturing at 37 ° C for 0, 1, 2, 3, 4, 6, 8 and 12 hours, the viral genomic RNA in the infected cells was recovered by thiocyanate guanidine lysis 25% ethanol precipitation. In the following, cDNA synthesis from RNA in each test method and PCR amplification method are shown in detail.

(Adsorption inhibition test and vRNA synthesis inhibition test of influenza virus)

In the adsorption inhibition test and the vRNA synthesis inhibition test, reverse transcription was performed from the recovered 0.5-5 μg RNA using Super Script III (Invitrogen) reverse transcriptase and T7 cRNA (1-12) primers according to the attached instructions. The reaction was carried out to synthesize cDNA of vRNA. PCR was amplified by using a set of oligonucleotide primers specific for the virus (Udorn) NP gene using the synthesized cDNA as a template. PCR was performed for 45 cycles (5 seconds, 95 degreeC, 34 second, 64 degreeC) using SYBR Premix taq polymeraseII (Invitrogen).

The copy number of the vRNA per each obtained hole is shown in FIG. The adsorption copy number at time 0 of the CSD3 untreated virus was 6.7 × 10 ⁸ / hole, whereas adsorption was suppressed to 2.2 × 10 ⁸ / hole and about 1/3 by 1 µg / ml CSD3 treatment. In addition, the amount of vRNA synthesis of the CSD3 untreated virus was 2.2 × 10 ¹⁰ / holes (12 hours after culture), whereas in the 1 µg / ml CSD3 treatment, the amount of vRNA synthesis was 3.2 × 10 ⁸ / holes, and the amount of vRNA synthesis was about 1/70. As reduced.

(mRNA synthesis inhibition test)

In the primary / secondary transcriptional inhibition test of virus by CSD3, reverse transcriptase reaction was carried out using reverse transcriptase and T7T (18) VN primers to synthesize cDNA of mRNA. PCR was amplified by using a set of oligonucleotide primers specific for NP gene using the synthesized cDNA as a template. PCR was performed for 45 cycles (5 second, 95 degreeC, 34 second, 64 degreeC) using SYBR Premix taq polymeraseII (Invitrogen).

The copy number of mRNA per each obtained hole is shown in FIG. The copy number of mRNA of CSD3 untreated virus was 2.6 × 10 ¹⁰ / holes (12 hours after incubation), whereas in 1 μg / ml CSD3 treatment, 4.3 × 10 ⁷ / holes and mRNA synthesis amount was about 1/600. Decreased.

(cRNA synthesis inhibition test)

In the replication step inhibition test of the virus by CSD3, reverse transcription was performed using a reverse transcriptase and a T7 vRNA (1-13) primer to synthesize cDNA of cRNA. PCR was amplified by using a set of oligonucleotide primers specific for NP gene using the synthesized cDNA as a template. PCR was performed for 45 cycles (5 second, 95 degreeC, 34 second, 64 degreeC) using SYBR Premix taq polymeraseII (Invitrogen). The copy number of the cRNA per each obtained hole is shown in FIG. The copy number of cRNA of the CSD3 untreated virus was 9.7 × 10 ⁶ / holes (after 8 hours of culture), whereas in 1 μg / ml CSD3 treatment, it was 2.5 × 10 ⁴ / holes, and the cRNA synthesis amount was about 1/400. Decreased.

Example 3

(Hemolysis inhibition test)

A sample stock solution was obtained by dissolving CSD3 in 5% ethanol (5 mg / ml). Influenza virus (10 HA to 320 HA) and CSD3 (10 ² to 10 ⁶ fold dilution of sample stock with TGS) were incubated at room temperature for 10 minutes. After the reaction, influenza virus treated with CSD3 and 100 µl of 10% chicken erythrocytes were mixed and maintained at room temperature for 30 minutes. Erythrocytes were collected by centrifugation and the precipitate was suspended in a buffer of pH 5.3 (136 mM NaCl, 2.68 mM KCl, 10 mM CH ₃ COONa). The suspension was incubated at 37 ° C. for 30 minutes and then centrifuged. The absorbance at 409 nm of the supernatant was measured, and the hemolytic activity by the addition of the Chinese quince was evaluated with the absorbance of the Chinese quince untreated virus as 1.

The obtained result is shown in FIG.

Example 4

CSD3 was added to the virus (16000 HA) and maintained at room temperature for 1 hour. Electron microscopy of the virus particles was carried out by a negative staining method with 2% uranium acetate.

These results are shown in FIG.

1 is an electron microscope image showing the disruptive activity of the quince extract (CSD3) against the viral envelope membrane, (a) shows the case where the quince extract is added, and (b) shows the case without addition of the quince extract.

Electron microscopy images of the virus treated with CSD3 showed an adhesion layer of about 2 nm wide CSD3 on the outside of the HA-NA spike on the surface of the particle, and there was a point of damage to the envelope, which allowed the dye to penetrate into the particle. The internal structure was visualized (FIG. 1 (a)).

(conclusion)

In the Chinese quince extract (CSD3) treated virus, virus proliferation was stopped at an earlier stage than primary transcription. Inactivation at the adsorption-membrane fusion step of the virus also appeared, but further, the synthesis of RNA was suppressed, suggesting that there was any defect in the process from membrane fusion to primary transcription. The damage to the envelope seen in the electron microscope may be the cause of this defect.

Using the Chinese quince extract prepared in Example 1, mouthwashes, inhalants, troches, sprays, chewing gums, candies, fruits, beverages, powders, tablets, water solubles, gummi jelly, chocolate, biscuits, ice, sherbet, Soup, jam, wet tissue and mask were prepared. The prescription is shown as an Example below.

Example 5

Prescription of mouthwash

Ethanol 2.0 wt%

1.0% by weight fragrance

Saccharin 0.05 wt%

0.01% by weight of chlorhexidine hydrochloride

CSD3 0.5 wt%

Water rest

100.0 wt%

Example 6

Prescription of inhalants

Ethanol 5.0 wt%

CSD3 1.0 wt%

Water rest

100.0 wt%

Example 7

Prescription of troche

Glucose 72.3 wt%

Lactose 19.0 wt%

Arabian rubber 6.0 wt%

1.0% by weight fragrance

0.7% by weight sodium monofluorophosphate

CSD3 1.0 wt%

100.0 wt%

Example 8

Prescription of Spray

Ethanol 25.0 wt%

Citric acid 1.5 wt%

Sodium citrate 1.0% by weight

CSD3 0.5 wt%

Water rest

100.0 wt%

Example 9

Chewing Gum Prescription

Gum base 20.0 wt%

54.7 wt% sugar

15.0% by weight glucose

Starch syrup 9.3 wt%

0.5% by weight fragrance

CSD3 0.2 wt%

100.0 wt%

Example 10

Candy prescription

50.0 wt% sugar

Starch syrup 34.0 wt%

Citric Acid 1.0 wt%

Fragrance 0.2 wt%

CSD3 0.4 wt%

Water rest

100.0 wt%

Example 11

Prescription

Sugar 76.1 wt%

Glucose 19.0 wt%

0.2% by weight sucrose fatty acid ester

Fragrance 0.2 wt%

CSD3 0.5 wt%

Water rest

100.0 wt%

Example 12

Prescription of drink

Orange Juice 30.00% by weight

15.24% by weight of isomerized

Citric Acid 0.10 wt%

Vitamin C 0.04 wt%

Perfume 0.10% by weight

CSD3 0.10 wt%

Water rest

100.00 wt%

Example 13

Powder prescription

Corn starch 55.0 wt%

Carboxy cellulose 40.0 wt%

CSD3 5.0 wt%

100.0 wt%

Example 14

Prescription of tablets

Lactose 70.0 wt%

15.0 wt% crystalline cellulose

Magnesium Stearate 5.0% by weight

CSD3 10.0 wt%

100.0 wt%

Example 15

Prescription of Hydrant

Ethanol 2.00 wt%

1.00% by weight

Saccharin 0.05 wt%

0.01% by weight of chlorhexidine hydrochloride

CSD3 0.50 wt%

Water rest

 100.00 wt%

Example 16

Gumi jelly prescription

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% by weight

CSD3 0.05 wt%

100.00 wt%

This application claims the priority from Japanese Patent Application No. 2009-218643 for which it applied on September 24, 2009, and Japanese Patent Application No. 2009-278462 for which it applied on December 8, 2009, The content is Is incorporated herein by reference.

Claims (9)

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. Influenza virus adsorption inhibitor, characterized in that the extract obtained by extracting the Chinese quince with 50% ethanol water, the extract obtained by column fractionation and purification of the extract. An inhibitor of virus mRNA synthesis in an influenza virus infected cell, wherein the extract is extracted with 50% ethanol water, and the extract obtained by column fractionation and purification of the extract is an active ingredient. An inhibitor of virus vRNA synthesis in influenza virus infected cells, wherein the extract is obtained by extracting the quince with 50% ethanol water, and extracting the purified extract by column fractionation. An inhibitor of viral cRNA synthesis in influenza virus infected cells, wherein the extract is obtained by extracting the Chinese quince with 50% ethanol water, and extracting the purified extract by column fractionation. A hemolytic inhibitor in a viral membrane fusion activity test, wherein the extract is obtained by extracting the Chinese quince with 50% ethanol water, and extracting the purified extract by column fractionation. An influenza virus envelope destroyer, wherein the Chinese quince is extracted with 50% ethanol water, and the extract obtained by column fractionation and purification of the extract is used as an active ingredient. Food and drink having anti-influenza virus action containing Chinese quince extract.

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