HK1171944A - Anti-influenza virus agent - Google Patents

Description

Anti-influenza virus agents

Technical Field

The present invention relates to an anti-influenza virus agent, an influenza virus adsorption inhibitor, a viral mRNA synthesis inhibitor in influenza virus-infected cells, a viral vRNA synthesis inhibitor in influenza virus-infected cells, a viral cRNA synthesis inhibitor in influenza virus-infected cells, a hemolysis inhibitor in a virus membrane fusion activity test, and an influenza virus envelope-disrupting agent, each of which contains a plant-derived extract having an influenza virus infection-inhibiting effect as an active ingredient.

Background

Influenza viruses that cause influenza epidemics every year are RNA viruses with a membrane envelope of about one ten thousandth of a millimeter in diameter. The antigen is classified into type A, type B and type C3 according to the difference of antigenicity, and the type A and type B are widely popular. On the surface of the particle of these viruses, 2 kinds of glycoproteins, Hemagglutinin (HA) and Neuraminidase (NA), are present as spike-like projections, and gene RNAs segmented into 8 pieces are present inside. HA and NA located on the surface of viruses are frequently mutated within the same subtype, and new antigenic mutants appear each year.

Influenza virus released by a cough droplet invades from the nose or mouth of a human being, is adsorbed to mucosal epithelial cells of the upper respiratory tract by spike glycoprotein HA on the surface layer of the virus, and starts to proliferate after invading into the cells. Through recent studies, the infection mechanism of viruses has been clarified. Viruses bind to receptors formed by sugar chains present on the surface layers of human target cells, are taken into endosomes, invade into cells by fusion between viral membranes and endosomal membranes, undergo uncoating and migration to start expression and replication of viral genes, and finally proliferate by forming progeny virions by budding from host cell membranes.

Several days since the infection with influenza virus, symptoms such as sudden fever, headache, arthralgia, and general weakness are manifested, and respiratory organ symptoms such as cough, throat pain, nasal discharge, and nasal obstruction are accompanied. Unlike the so-called cold, it is characterized by a strong infectivity, triggering an explosive epidemic in a short time. In addition, the structure of the HA protein of influenza virus is mutated repeatedly every year, and the antibody produced by past infections HAs little effect and is also a main cause of infection spread.

In order to suppress infection with influenza virus, inhibition of adsorption to epithelial cells, inhibition of cell invasion, inhibition of gene transcription and replication, inhibition of protein synthesis, inhibition of release from cells, and the like are considered, and each of these is a target of antiviral drugs. Antiviral agents such as amantadine, rimantadine, zanamivir have been developed so far, but side effects such as allergy, psychoneurosis, digestive system symptoms, autonomic nervous system symptoms have been reported, and much attention has been paid to their application.

Furthermore, since influenza virus infects and proliferates in the respiratory mucosal epithelium and the current year's epidemic cannot be predicted accurately, it is considered difficult to suppress infection by vaccination. Frequent gargling, avoidance of dryness of the throat, adequate nutrition and rest, etc. are considered to be the most effective preventive measures at present. It is expected to develop an anti-influenza virus agent which has a good infection-suppressing effect and is safe and can be used on a daily basis.

In recent years, polyphenol components of tea or black tea have been reported as anti-influenza materials derived from natural products (non-patent documents 1 and 2), and human tests have shown that gargling with black tea can inhibit actual viral infection (non-patent document 3). Further, it has been reported that a flavonoid component derived from scutellaria baicalensis exhibits an influenza infection inhibitory effect by the sialidase inhibitory activity of a virus (non-patent document 4). In addition, antiviral effects of a herbal preparation such as a Japanese diebi decoction of cassia twig (patent document 1), a blackcurrant extract (patent document 2), a anthocyanin of potato (patent document 3), a guava leaf extract (patent document 4), and an apocynum venetum extract (patent document 5) have been reported.

Further, anti-influenza agents containing extracts of flower buds or flower petals of rosaceous plants as active ingredients have been disclosed for rosaceous plants (patent document 6), and anti-influenza virus effects of extracts of chaenomeles speciosa (patent document 8) and column-chromatography isolates of chaenomeles speciosa (non-patent document 5) have been reported. However, reports on the inhibitory effect on the adsorption of influenza virus, the inhibitory effect on the synthesis of viral mRNA in influenza virus-infected cells, the inhibitory effect on the synthesis of viral vRNA in influenza virus-infected cells, the inhibitory effect on the synthesis of viral cRNA in influenza virus-infected cells, the inhibitory effect on hemolysis in the test of viral membrane fusion activity, and the inhibitory effect on the envelope disruption of influenza virus, which are shown in the present patent, have not been found, and are disclosed for the first time by the present invention.

Prior art documents

Patent document

Patent document 1: japanese patent laid-open No. 6-199680

Patent document 2: japanese patent laid-open No. 2000-212092

Patent document 3: japanese patent laid-open No. 2001-316399

Patent document 4: japanese patent laid-open No. 2000-273048

Patent document 5: japanese patent laid-open No. Hei 11-71296

Patent document 6: japanese patent laid-open No. 2002-145790

Patent document 7: japanese patent application laid-open No. 2002-020305

Patent document 8: japanese patent laid-open No. 2005-343836

Non-patent document

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: J.Infectionology, 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-

Summary of the invention

Technical problem to be solved by the invention

The purpose of the present invention is to provide an influenza virus adsorption inhibitor, a viral mRNA synthesis inhibitor in influenza virus-infected cells, a viral vRNA synthesis inhibitor in influenza virus-infected cells, a viral replication inhibitor in influenza virus-infected cells, a hemolysis inhibitor in a viral membrane fusion activity test, and an influenza virus envelope-disrupting agent, using a plant extract which is highly safe and can be used on a daily basis.

Means for solving the problems

In order to solve the above problems, the present inventors focused on papaya having no side effects and high safety, and found the effect of papaya extract by RT-PCR analysis of mRNA, vRNA, cRNA in MDCK cells to which influenza virus was added, hemolytic reaction using chicken red blood cells, and electron microscope observation of virus particles, thereby completing the present invention.

That is, the present invention provides an influenza virus adsorption inhibitor, a viral mRNA synthesis inhibitor in influenza virus-infected cells, a viral vRNA synthesis inhibitor in influenza virus-infected cells, a viral cRNA synthesis inhibitor in influenza virus-infected cells, a hemolysis inhibitor in a virus membrane fusion activity test, and an influenza virus envelope-disrupting agent, each of which is characterized by using a papaya (Chaenomeles sinensis, pseudococcus) extract as an active ingredient. The present invention also provides a food or drink having an anti-influenza virus activity, which contains the papaya extract.

ADVANTAGEOUS EFFECTS OF INVENTION

The present invention provides an influenza virus adsorption inhibitor, a viral mRNA synthesis inhibitor in influenza virus-infected cells, a viral vRNA synthesis inhibitor in influenza virus-infected cells, a viral replication inhibitor in influenza virus-infected cells, a hemolysis inhibitor in a virus membrane fusion activity test, and an influenza virus envelope-disrupting agent, each of which comprises a highly safe papaya extract as an active ingredient and has a strong effect on influenza viruses. Further, since the papaya extract as an active ingredient of the present invention is highly safe, it can be widely used in daily life as an influenza virus infection-inhibiting product by adsorption, impregnation, and addition to a mask, an air conditioner filter, clothes, a wet tissue, a spray solution, and the like. In addition, the extract can be added to foods and beverages such as chewing gum, candy press, beverage, etc., and can be daily used and taken as a food and beverage having an anti-influenza virus effect. The present invention is effective for the prevention of infection with influenza virus and the treatment of diseases caused by influenza virus.

Brief description of the drawings

FIG. 1 shows the destructive activity of papaya extract on viral envelopes.

FIG. 2 shows the adsorption inhibitory activity, vRNA synthesis inhibitory activity, mRNA synthesis inhibitory activity and cRNA synthesis inhibitory activity of papaya extracts.

FIG. 3 shows the hemolytic inhibitory activity of papaya extract.

Modes for carrying out the invention

Among the pawpaw as the raw material of the present invention, the fruit thereof is preferably used.

The method for obtaining the extract of the present invention from the pulverized product of the above-mentioned plant is not particularly limited, and 1 or 2 or more kinds of mixed solvents of water, lower alcohols such as methanol, ethanol, n-propanol and n-butanol, and organic solvents such as ether, ethyl acetate, acetone, glycerol, and propylene glycol are added and extracted by a conventionally employed extraction method. However, if it is considered that the anti-influenza virus agent of the present invention is orally ingested, it is preferable to extract with ethanol or a mixture thereof from the viewpoint of safety.

The extraction conditions are not particularly limited, but are preferably about 1 to 5 hours at 50 to 90 ℃. The extract is filtered and the extraction solvent is distilled off, and then concentrated under reduced pressure or freeze-dried, and the obtained product can be used. Further, a product obtained by separating and purifying these extracts by organic solvent partition, column chromatography, or the like may also be used.

The form of use of the present invention is not particularly limited, and the plant extract as an example of the active ingredient may be added with a solvent, a dispersant, a carrier for preparation, an emulsifier, a diluent, a stabilizer, etc. to prepare any preparation such as powder, tablet, troche, inhalant, mouthwash, gargle, suppository, injection, etc., and the administration route may be oral administration, respiratory administration, intravenous administration, rectal administration, subcutaneous administration, intradermal administration, etc. In this case, the dose to be administered to an adult 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, and is preferably 0.001% by weight or more, more preferably about 0.01% by weight or more.

Further, the present invention can provide an infection-suppressing article which is useful for preventing influenza by adsorbing, impregnating, and adding to a mask, an air-conditioning filter, clothes, a wet tissue, a spray solution, or the like. The amount of the plant extract adsorbed and added for these uses varies depending on the form of the infection control product, and is not always clear, but is preferably 0.001 to 5% by weight.

Further, since the present invention is highly safe, the following foods and beverages can be incorporated and used in daily life: such as snacks such as chewing gum, candy, pressed candy, soft candy, chocolate and biscuit, cold drinks such as ice cream and ice cream bar, drinks, soup, jam and the like. The amount of the additive is preferably 0.001 to 5% by weight, more preferably about 0.01 to 1% by weight, based on the food or drink, depending on the form of use and taste of the extract.

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

[ example 1]

(preparation of papaya extract)

The dried and crushed pawpaw is extracted by 50% EtOH and separated by Diaion HP-20 to obtain 5 separated parts (CSD 1-5). The most active of these fractions, CSD3 (40% EtOH-eluting fraction), was used as a sample.

That is, a flask to which 300g of dried papaya fruits and 3000ml of 50% ethanol were added was equipped with a reflux condenser, and extraction was performed while refluxing for 1 hour. The resulting extract was separated by filtration, the solvent was removed, and freeze-dried to obtain 69g of an extract.

The above papaya 50% ethanol extract was applied to Diaion HP-20 (manufactured by Mitsubishi chemical corporation) column. Eluting with water, 20% ethanol, 40% ethanol, and 60% ethanol in this order. Fractions eluted by the ethanol concentration 40% aqueous solution (CSD3) were collected. The yield of this isolated fraction was about 8.6g on a dry weight basis. The product contains 54% of phenol obtained by vanillin-hydrochloric acid method [ with (-) -epicatechin as standard ], and contains mainly phenolic substances.

[ example 2]

The CSD 3-based effect of inhibiting viral adsorption, the effect of inhibiting vRNA synthesis, the effect of inhibiting mRNA synthesis, and the effect of inhibiting cRNA synthesis were analyzed by RT-PCR method by extracting viral RNA. CSD3 was dissolved in 50% ethanol under conditions of 50mg/ml, and the resulting solution was used as a sample stock solution. The sample stock solution was diluted appropriately (dilution 10) with Tris-glucose physiological saline (TGS)²To 10⁶Duplicate), 50. mu.l of these sample dilutions were mixed with 50. mu.l of virus solution (4X 10)⁴To 4X 10⁶PFU (plaque forming unit)/ml) were mixed and reacted at room temperature for 10 minutes. 0.1ml of the above sample was inoculated into monolayer culture (6-well plate) of Madin-Darby Canine Kidney (MDCK) cells supplemented with 0.4ml of TGS, the virus was adsorbed at room temperature for 30 minutes, the supernatant was removed after the reaction, 0.5ml of the culture medium was added, and after 0, 1, 2, 3, 4, 6, 8, and 12 hours of culture at 37 ℃, viral genomic RNA in infected cells was recovered by a 25% ethanol precipitation method using guanidinium thiocyanate lysis. To eachThe cDNA synthesis method and PCR amplification method from RNA in the test method will be described in detail.

(adsorption inhibition test and vRNA synthesis inhibition test for influenza Virus)

In the adsorption inhibition test and the vRNA synthesis inhibition test, 0.5 to 5. mu.g of RNA recovered was subjected to reverse transcription reaction using a Super Script III (Invitrogen corporation, インビトロジエン)) reverse transcriptase and a T7cRNA (1-12) primer according to the attached instructions to synthesize vRNA cDNA. The synthesized cDNA was used as a template for PCR amplification using 1 set of oligonucleotide primers specific for the NP gene of virus (Udorn). PCR was carried out for 45 cycles (5 sec, 95 ℃ C.; 34 sec, 64 ℃ C.) with SYBR Premix taq polymerase II (Invitrogen).

The copy number of vRNA obtained for each well is shown in figure 2. The 0 hour adsorbed copy number of CSD3 untreated virus was 6.7X 10⁸Per well and 2.2X 10 after treatment with 1. mu.g/ml of CSD3⁸Pore, adsorption was suppressed to about 1/3. Furthermore, the vRNA synthesis amount of the CSD3 untreated virus was 2.2X 10¹⁰Perwell (after 12 hours of culture) and 3.2X 10 after treatment with 1. mu.g/ml of CSD3⁸Per well, vRNA synthesis decreased to about 1/70.

(mRNA synthesis inhibition test)

In the primary/secondary transcription inhibition test of the virus based on CSD3, cDNA of mRNA was synthesized by reverse transcription using reverse transcriptase and T7T (18) VN primer. The synthesized cDNA was used as a template, and PCR amplification was performed using 1 set of oligonucleotide primers specific for the NP gene. PCR was performed with SYBR Premixtaq polymerase II (Invitrogen) for 45 cycles (5 sec, 95 ℃ C.; 34 sec, 64 ℃ C.).

The copy number of the mRNA obtained for each well is shown in FIG. 2. The amount of mRNA synthesized by CSD3 untreated virus was 2.6X 10¹⁰Perwell (after 12 hours of culture) and 4.3X 10 after treatment with 1. mu.g/ml of CSD3⁷Per well, the amount of mRNA synthesized was reduced to about 1/600.

(cRNA synthesis inhibition assay)

CSD 3-based virusesIn the replication stage inhibition test of (3), a reverse transcription reaction was performed using a reverse transcriptase and a T7vRNA (1-13) primer to synthesize cRNA cDNA. The synthesized cDNA was used as a template, and PCR amplification was performed using 1 set of oligonucleotide primers specific for the NP gene. PCR was performed with SYBRPremix taq polymerase II (Invitrogen) for 45 cycles (5 sec, 95 ℃ C.; 34 sec, 64 ℃ C.). The copy number of the cRNA obtained for each well is shown in fig. 2. The cRNA synthesis amount of the CSD3 untreated virus was 9.7X 10⁶Perwell (after 8 hours of culture) and 2.5X 10 after treatment with 1. mu.g/ml of CSD3⁴Per well, the amount of cRNA synthesized was reduced to about 1/400.

[ example 3]

(hemolysis inhibition test)

CSD3 was dissolved in 5% ethanol (5mg/ml) to prepare a sample stock solution. Influenza virus (10HA to 320HA) and CSD3 (sample stock was diluted 10 with TGS)²To 10⁶Fold) was incubated at room temperature for 10 minutes. After the reaction, 100. mu.l of 10% chicken red blood cells were mixed with the influenza virus treated with CSD3 and the mixture was kept at room temperature for 30 minutes. The red blood cells were collected by centrifugation, and the pellet was suspended in a buffer solution (136mM NaCl, 2.68mM KCl, 10mM CH) at pH5.3₃COONa). The suspension was incubated at 37 ℃ for 30 minutes and centrifuged. The absorbance of the supernatant at 409nm was measured, and the hemolytic activity when papaya was added was evaluated with the absorbance of papaya untreated virus being 1.

The results obtained are shown in FIG. 3.

[ example 4]

CSD3 was added to the virus (16000HA) and kept at room temperature for 1 hour. The electron microscopic observation of the virus particles was performed by a negative staining method using 2% uranium acetate.

These results are shown in FIG. 1.

FIG. 1 is an electron microscope image showing the activity of papaya extract (CSD3) to destroy viral envelopes.

In an electron microscope image of the CSD 3-treated virus, an adhesion layer considered to be CSD3 with a width of about 2nm was observed on the outer side of the spikes of HA and NA on the particle surface, and the damaged part was enveloped, and the inside of the particle was permeated with a stain to visualize the internal structure (fig. 1 (a)).

(conclusion)

Papaya extract (CSD3) treated virus showed a cessation of viral proliferation at a stage prior to primary transcription. Further, inactivation of the virus at the stage of adsorption and membrane fusion was observed, and RNA synthesis was suppressed to a greater extent, suggesting that there was some defect associated with the process of membrane fusion to primary transcription. Damage to the envelope as exhibited by electron microscopy may be the cause of the defect.

Using the papaya extract prepared in example 1, mouthwash, inhalant, troche, spray, chewing gum, candy, pressed candy, beverage, powder, tablet, gargle, soft candy, chocolate, biscuit, ice cream, soup, jam, wet tissue, and mask were prepared. The following examples show the formulations.

[ example 5]

Formula of mouthwash

[ example 6]

Formulation for inhalant

[ example 7]

Tablet-containing formulation

[ example 8]

Formula of spray liquid

[ example 9]

Chewing gum formulation

[ example 10]

Formula of candy

[ example 11]

Formula of tablet candy

[ example 12]

Beverage formula

[ example 13]

Formulation of powder

[ example 14]

Formulation of tablets

[ example 15]

Formula of gargle

[ example 16]

Formula of soft sweet

The present application claims priority based on japanese patent application No. 2009-.

Claims (9)

1. An anti-influenza virus agent characterized by comprising an extract of papaya (Chaenomeles sinensis, pseudococcus sinensis) as an active ingredient.

2. An anti-influenza virus agent characterized by comprising extracting papaya with a 50% aqueous ethanol solution, purifying the extract by column chromatography, and using the obtained extract as an active ingredient.

3. An influenza virus adsorption inhibitor characterized by comprising extracting papaya with a 50% aqueous ethanol solution, purifying the extract by column chromatography, and using the obtained extract as an active ingredient.

4. A viral mRNA synthesis inhibitor in influenza virus-infected cells, characterized in that papaya is extracted with a 50% aqueous ethanol solution, and the extract is purified by column chromatography to obtain an extract as an active ingredient.

5. A viral vRNA synthesis inhibitor in influenza virus-infected cells, characterized in that papaya is extracted with a 50% aqueous ethanol solution, and the extract is purified by column chromatography to obtain an extract as an active ingredient.

6. A viral cRNA synthesis inhibitor in influenza virus-infected cells, characterized in that papaya is extracted with a 50% aqueous ethanol solution, and the extract is purified by column chromatography to obtain an extract as an active ingredient.

7. A hemolytic inhibitor in a viral membrane fusion activity test, characterized in that papaya is extracted with a 50% aqueous ethanol solution, and the extract is purified by column chromatography to obtain an extract as an active ingredient.

8. An agent for disrupting an envelope of an influenza virus, characterized in that papaya is extracted with a 50% aqueous ethanol solution, and the extract is purified by column chromatography to obtain an extract as an active ingredient.

9. A food or drink characterized by containing a papaya extract and having an anti-influenza virus activity.