TW201125571A - Anti-influenza virus agent - Google Patents

Abstract

Disclosed are an inhibitor of the adsorption of an influenza virus, an inhibitor of the synthesis of viral mRNA in influenza virus-infected cells, an inhibitor of the synthesis of viral vRNA in influenza virus-infected cells, an inhibitor of the synthesis of viral cRNA in influenza virus-infected cells, an anti-hemolytic agent for use in a viral membrane fusion activity test, and an agent for disrupting an envelope of an influenza virus, each of which utilizes a plant extract that is highly safe for use on a daily basis. Specifically disclosed is an anti-influenza virus agent characterized by containing, as an active ingredient, an extract produced by extracting quince with a 50% aqueous ethanol, fractionating the resulting extract on a column, and purifying the fractionated product.

Description

201125571 VI. Description of the Invention: [Technical Field] The present invention relates to an anti-influenza virus agent, an influenza virus adsorption inhibitor, an infection, which has an extract of a plant having an action of inhibiting the infection of influenza virus as an active ingredient. Influenza virus cell virus mRN A synthesis inhibitor, influenza virus cell virus vRN A synthesis inhibitor, influenza virus cell infection virus c RNA synthesis inhibitor, virus membrane fusion activity test hemolytic inhibitor And a film destroyer for influenza viruses. [Prior Art] The influenza virus that causes influenza prevalence every year is an RN A virus having a diameter of about one ten thousandth of a millimeter and having a mantle. Because of their different antigenicity, they can be classified into three types: A, B, and C, while those with popular spread are type A and type B. On the surface of these virions, there are two kinds of glycoproteins, namely hemagglutinin (HA) and neuraminidase (ΝΑ), which are spiny-like, and there are 8 RNA gene fragments inside. The surface of the virus is frequently mutated in the same subtype, and new antigenic variants appear every year. The influenza virus contained in the droplets ejected during coughing invades the human body from the mouth or nose. The ratar epithelial cells of the upper respiratory tract are formed by the ratchetous glycoprotein HAW on the surface of the virus. After invading the cells, the virus begins to proliferate. According to recent studies, it has been clearly understood that the virus is infected. The virus system binds to the receptor 5-201125571 (receptor) composed of a sugar chain on the surface of the target cell existing in human, and then invades the cell by fusion of the virus membrane and the endosome membrane, and is subjected to shelling. - Transfer, expression and replication of viral genes, and finally release from the host cell membrane by budding to form new viral progeny particles and proliferate. Due to the infection with the influenza virus, sudden fever, headache, joint pain, general fatigue and other symptoms appear within a few days, and coughing or sore throat, runny nose, nasal congestion and other respiratory symptoms may occur before and after. That is to say, unlike the common cold, it is characterized by strong infection and causing an explosive epidemic in a short period of time. In addition, the HA protein structure of the influenza virus recurs every year, and the antibodies produced by past infections have little effect and are the main cause of the spread of infection. In order to suppress the infection of influenza virus, it is considered that it is necessary to hinder its adsorption to epithelial cells, hinder its invasion into cells, inhibit transcription and replication of viral genes, hinder protein synthesis, inhibit its release from cells, etc., and become antiviral drugs, respectively. The goal. Antiviral drugs such as amantadine, rimantadine, and zanamivir have been developed to date, but side effects such as allergies, neuropsychiatric symptoms, digestive symptoms, and autonomic nervous system symptoms have also been reported, and special attention should be paid to their use. Since influenza virus is infected and proliferated in the respiratory mucosal epithelium and cannot correctly predict the epidemic of the year, it is generally considered difficult to suppress infection by vaccination. Frequent use of tinctures, avoiding dry throat and adequate intake of nutrients and rest are currently considered to be the most effective preventive measures. We hope to develop anti-influenza virus agents that have high anti-infection effects, and thus have no safety concerns, and can be used in daily life. -6- 201125571 In recent years, 'the polyphenols of green tea and black tea from natural substances have been reported (non-patent documents! and 2), and it has been clearly confirmed by human trials that black tea liquid is used for bismuth. In fact, it can suppress viral infection (Non-Patent Document 3). Further, the flavonoid component derived from Astragalus has been reported to have an effect of suppressing influenza infection by inhibiting the sialidase activity of the virus (Non-Patent Document 4). Further, the traditional Chinese medicine preparation, Keishinieppiichito (patent document 1), black currant extract (patent document 2), potato anthocyanin (patent document 3), guava leaf extract (patent document 4) Apocynum venetum extract (Patent Document 5) and the like are reported to have antiviral effects. In addition, it has also been disclosed that in the Rosaceae plant, the anti-influenza agent with its flower bud or petal extract as an active ingredient (Patent Document 6), the molded extract (Patent Document 8), and the pipe-column liquid separation (Non-Patent Document 5) Anti-influenza virus effect. However, it has not been found that the column-column liquid substance shown in the patent application has the effect of inhibiting the influenza virus adsorption effect of the virus infecting influenza virus cells, and infecting influenza virus cells. The virus vRNA synthesis inhibitory effect, the CRN A synthesis inhibitory effect of the virus infected with influenza virus cells, the hemolysis inhibitory effect of the virus membrane fusion activity test, and the report on the destruction effect of the influenza virus virus are first explained in this case. . [Prior Art Document] [Patent Document 1] Japanese Patent Laid-Open Publication No. Hei No. Hei. No. Hei. No. Hei. No. Hei. No. 2001-316. [Patent Document 5] Japanese Laid-Open Patent Publication No. Hei No. Hei. No. 2002-145790 [Patent Document 7] JP-A-2002-020305 [Patent Document 8] Kokusai 2〇〇5-3 43 8 3 6 [Non-Patent Document] [Non-Patent Document 1] Journal of Infectious Diseases, 68 ( 7 ) 824-829 ( 1 994 ) [Non-Patent Document 2] Infectious Diseases Journal, 70 ( 1 1 ) 1 1 90-1 1 92 (1 996 ) [Non-Patent Document 3] Journal of Infectious Diseases, 7 1 ( 6 ) 487-494 ( 1 997 ) [Non-Patent Document 4] Chem. Pharm Bull. 38 (5) 1329-1332 (1990) [Non-Patent Document 5] Journal of Ethnopharmacology, 118, 108-112 (2008) [Summary of the Invention] [Problems to be Solved by the Invention] The object of the present invention is to use daily A plant extract that is safe to use and safe, provides influenza virus adsorption inhibitors, viral mRNA synthesis inhibitors that infect influenza virus cells, and infects influenza virus The extracellular virus vRNA synthesis inhibitors, 201,125,571 cells infected with influenza virus of viral replication inhibitors, viral membrane fusion activity of the test agent inhibition of hemolysis and membrane damage influenza virus agent sets. [Means for Solving the Problem] In order to solve the above-mentioned problems, the present inventors focused on the analysis of mRNA and vRNA 'cRNA of MDCK cells to which influenza virus was added, and the RT-PCR analysis of the mRNA and the like. The use of the hemolytic reaction of chicken red blood cells, the observation of the virions by electron microscopy, and the effect of the chess extracts, the present invention is completed. That is, the present invention is characterized by an influenza virus adsorption inhibitor having an extract of Chaenomeles sinensis (Pseudocydonia sinensis) as an active ingredient, a viral mRNA synthesis inhibitor for infecting influenza virus cells, and influenza infection. Virus vRNA synthesis inhibitor of viral cells, virus CRN A synthesis inhibitor of influenza virus-infected cells, hemolytic inhibitor of virus membrane fusion activity test, and film breaker of influenza virus. Further, the present invention is a food or drink containing a mold-extracted extract having an anti-influenza virus action. [Effects of the Invention] The present invention provides an influenza influenza virus adsorption inhibitor having a strong effect on influenza virus and a virus mRNA synthesis inhibition of influenza virus cells by using a highly safe chess extract as an active ingredient. Agent, virus vRNA synthesis inhibitor for influenza virus cells, virus replication inhibitor for influenza virus cells, virus membrane fusion-9 - 201125571 Hemolytic inhibitor for activity assay and membrane destroyer for influenza virus . In addition, since the active ingredient of the present invention has high safety, it can be widely used in daily life by adsorbing, impregnating, adding it to a mask, an air conditioner screen, clothes, a wet tissue, a spray liquid, and the like. As an anti-infective influenza virus product. Further, it is added to a food such as chewing gum, candy, ingot, or dip, and can be used daily for ingestion of a food having an anti-flucy virus effect. The present invention is effective for the prevention of infection by influenza virus or the treatment of diseases caused by influenza virus. It is preferred that the raw material of the present invention is molded to use the fruit thereof. The method for obtaining the extract of the present invention from the crushed material of the above plant is not particularly limited, and a lower alcohol such as water, methanol, ethanol, n-propanol or n-butanol, ether, methyl acetate or acetone may be added. One or a mixture of two or more organic solvents such as glycerin and propylene glycol is extracted according to a conventional extraction method. However, when the anti-influenza virus of the present invention is orally administered, it is preferred to use water, ethanol or a mixture thereof for extraction from the viewpoint of a safe surface. The stroke conditions are not particularly limited to 5 0 to 90 ° C, preferably about 1 to 5 hours. After extracting the extract and removing the extraction solvent, the obtained product is concentrated or freeze-dried under reduced pressure. Further, it is also possible to use a product obtained by liquid separation and purification of the extracts by organic solvent separation, column chromatography or the like. The form of use of the product of the present invention is not particularly limited, and an exemplary plant extract obtained as an active ingredient may be added to a solvent, a dispersant, or a preparation, a carrier, an emulsifier, a diluent, a stabilizer, or the like. It is prepared into a preparation, a throat lozenge, an inhalant, an expectorant, a mouthwash, a suppository, and any preparation. The administration route can be administered by oral administration, respiratory administration, intravenous administration, rectal administration, subcutaneous administration, and skin. Intramedicine and so on. The dosage is preferably from 10 to 2000 mg/day for each extract, but is determined by the number. The amount of the various preparation extracts to be added is different depending on the preparation, but it is preferably added in an amount of 0.001% by weight or more, and more preferably in a ratio of about 重 〇 重. Further, by adsorbing, impregnating, or adding the present invention to a mask, a net, a clothes, a wet tissue, a spray liquid, or the like, a product which can be expected to be pre-K-infected with influenza is provided. In the use of these articles, the adsorption and addition amount of the planting shirt is determined by the manner in which the form of the infectious article should be suppressed, but it is added in a ratio of 0.001 to 5% by weight, and the safety of the present invention is additionally added. It can be mixed with snacks such as mouth 1 , ingots, soft candy, chocolate, biscuits, etc., ice cream, β water and other ice products, such as soup, soup and jam, can be used in different ways. The amount of addition varies depending on the form of use and the taste of the extract, and the ratio of the food and drink is preferably 0.01 to 5% by weight, and the ratio of about 〇·0 1~ is more preferable. [Embodiment] [Examples] Hereinafter, the present invention will be specifically described by way of examples and experimental examples, but it is not preferable to administer a drug or an injectable agent to an adult, and it is not preferable to use an air-conditioning filter inhibitory extract of t% or more. The confectionery jelly is invented at a relative weight of 1% by weight and -11 - 201125571 is not limited to these examples. [Example 1] (Modulation of the plant extract) After the mold-dried dried ground material was extracted with 50% EtOH, liquid separation was carried out using DIAION HP-20 to obtain 5 liquid separation materials (CSD 1 to 5). The most active CSD3 (40% EtOH dissolution fraction) in the aliquot was used as a sample. 300 g of the dried fruit product and 300 ml of 50% ethanol were also added to the flask, and the device was further cooled to a reflux for 1 hour. After filtering the obtained extract and removing the solvent, 69 g of an extract was obtained by freeze-drying. The above-mentioned 50% ethanol extract was dissolved in water, 20% ethanol, 40% ethanol, and 60% ethanol using a DIAION HP-20 column (manufactured by Mitsubishi Chemical Corporation). A component (CSD3) which was eluted with an aqueous solution having a concentration of 40% ethanol was used. The dry weight of the fraction of the liquid fraction was about 8.6 g. This product is based on the vanillin-hydrochloric acid method, and the phenol content is 54% [(-) epicatechin as a standard], and it is confirmed that it mainly contains an inert substance. [Example 2] The virus adsorption effect by C S D 3 , the ν R Ν Α synthesis inhibitory effect, the m R N A synthesis inhibitory effect, and the c R N A synthesis inhibitory effect were extracted by extracting the virus RN A and then analyzed by the RT-P CR method. A solution having a concentration of 50 mg / ml after dissolving C S D 3 in 50% ethanol was used as a sample stock solution. The test solution 201125571 is appropriately diluted with Tris-Glucose-Saline (TGS) (diluted 102 to 106 times), and 50 μl of the sample dilution is taken, and 50 μl of the virus solution (4χ104 to 4x106 PFU (plaque forming unit) / m 1 ) Mix and allow to react at room temperature for 10 minutes. The 0.1 ml of the liquid was further inoculated into a monolayer culture (6-well plate) of Madin-Darby canine kidney (MDCK) cells to which 0.4 ml of TGS was added, and the virus was adsorbed at room temperature for 30 minutes, after the reaction. The supernatant was removed, and the culture solution of 0 · 5 m 1 was added. After culturing at 1, 7 ° C for 1, 2, 3, 4, 6, 8 and 12 hours, the guanidine thiocyanate was dissolved. 25% ethanol precipitation method to recover viral genomic RNA in infected cells. The cDN A synthesis method and the PC R amplification method by RN A in each test method are shown in detail below. (Influenza virus adsorption inhibition test and vRNA synthesis inhibition test) In the adsorption inhibition test and the v RN A synthesis inhibition test, from the recovery of 0.5 to 5 pg of RNA, follow the instructions in the attached manual, using Super Script III (Invitrogen) Reverse transcriptase and T7 cRNA (1-1 2) primers were used to carry out reverse transcription reaction to synthesize cDNA of vRNA. Further, using the synthesized cDNA as a template, PCR amplification was carried out by a specific set of oligonucleotide primers of the virus (Udorn) NP gene. The PCR system was subjected to 45 cycles (5 seconds '95 ° C, 34 seconds, 64 ° C) using SYBR Premix taq polymerase II (Invitrogen). The gene copy number (COpy number) of each of the obtained v-wells is shown in Fig. 2 . Compared with the virus treated without CSD 3 at the third hour of adsorption-13- 201125571, the viral gene replication number was 6.7 χ 108/well, and the virus was adsorbed by 2.2×1 08/well with lpg/ml of CSD3. It is suppressed to about 1/3. In addition, the amount of synthesis of viral VRNAs treated with no CSD3 was 2.2 x 1 〇 10 / well (after 12 hours of culture), and the amount of synthesis of vRNA was reduced to about 3.2 x 108 / wells with CSD3 at lug / ml. 1/70. (mRNA synthesis inhibition test) In the virus primary/secondary transcription inhibition assay by CSD3, reverse transcription reaction was carried out using reverse transcriptase and T7T (18) VN primer to synthesize cDNA of mRNA. Further, using the synthesized cDNA as a template, PCR amplification was carried out by using a specific set of oligonucleotide primers of the ΝΡ gene. The PCR system was subjected to 45 cycles (5 seconds, 95 ° C, 34 seconds, 64 ° C) using SYBR Premix taq polymerase II (Invitrogen). The number of copies of the obtained mRNA of each well is shown in Fig. 2 . The number of copies of the mRNA relative to the virus not treated with CSD3 was 2·6χ101()/well (after 12 hours of culture), and 4.3Χ107/well was treated with CSD3 at lpg/ml, and the amount of mRNA synthesis was reduced to about 1/ 600. (cRNA synthesis inhibition test) In the virus replication phase inhibition assay by C S D 3 , reverse transcription reaction was carried out using a reverse transcription enzyme and a T7vRNA (1 -1 3 ) primer to synthesize a cDNA of cRNA. Further, using the synthesized cDNA as a template, PCR amplification was carried out by using a specific primer set of the NP gene. The PCR system used SYBR Premix taq polymerase II (Invitrogen) to enter 45 -14 - 201125571 cycles (5 seconds, 95 °C, 34 seconds, 64 °C). The number of copies of each of the obtained wells cRN A is shown in Fig. 2 . The number of copies of cRNA relative to virus cRNA not treated with CSD3 was 9.7xl〇6/well (cultured Μ, after-time), 2.5×104/well was treated with BSD/ml of CSD3, and the amount of cRNA synthesis was reduced to about 1/ 400 〇 [Example 3] (Blood inhibition test) A sample solution was prepared by dissolving CSD3 in 5% ethanol (5 mg/ml). Influenza virus (10HA to 320HA) and CSD3 (sample solution diluted 1 〇2 to 1 〇 6 times with T G S) were incubated for 1 室温 at room temperature. After the reaction, the CSD3-treated influenza virus was mixed with 10% chicken red blood cell ΙΟΟμΙ and kept at room temperature for 30 minutes. The red blood cells were aggregated by centrifugation, and then suspended in a buffer of ρΗ5·3 (1 36 mM NaCl, 2.6 8 mM KC1, 10 mM CH3COONa). The suspension was further incubated at 37 ° C for 30 minutes and centrifuged. The absorbance of the supernatant at 409 ηπ] was measured, and the absorbance of the virus which was not used for the planting was 1, and the evaluation of the hemolytic activity by the addition of the chess was performed. The results obtained are shown in Fig. 3. [Example 4] CSD3 was added to the virus (16,000 Torr) and kept at room temperature! hour. Observation of virions by electron microscopy was carried out by negative staining using 2% uranyl acetate. -15- 201125571 These results are shown in Figure 1. Fig. 1 is an electron micrograph showing the destruction activity of the molded extract (CSD3) on the viral envelope, and (a) shows the case where the addition of the chess extract is shown, where (b) indicates that the molded extract is not added. In the electron micrograph of the virus treated with CSD3, an adhesion layer of CSD3 about 2 nm wide on the outer side of the HA.NA spike on the surface of the particle was observed, and the lesion was visible on the cover film, and the stain was soaked. Inside the particle, the internal structure is visualized (Fig. 1 (a)). (Conclusion) The virus treated with the modeled extract (CSD3) showed that the virus proliferation was stopped at the earlier stage of the primary transcription. It also showed activity in the adsorption of the virus • membrane fusion phase, but showed more inhibition of RN A synthesis, suggesting a related defect in the process from membrane fusion to primary transcription. Membrane damage exhibited by an electron microscope may be the cause of this defect. Using the molded extract prepared in Example 1, the gargle, inhalant, throat lozenge, spray liquid, chewing gum, candy, ingot, drink, powder, lozenge, mouthwash, jelly, Chocolate, biscuits, ice cream, frozen juice, soup, jam, wet wipes, masks. The formulation is exemplified below by way of examples. -16- 201125571 [Example 5] Antimony formula Ethanol flavor saccharin Hydrochlorhexidine hydrochloride CSD3 Water 2.0% by weight 1 .0 wt% 0.0 5 wt% 〇. 〇1 wt% 0.5 wt% balance 1 0 0.0 wt% [Example 6] Inhalant Formulation Ethanol CSD3 Water 5.0% by weight 1.0% by weight Balance 1 0 0.0% by weight [Example 7] Throat ingot formula Glucose lactose gum arabic spice sodium monofluorophosphate CSD3 7 2.3% by weight 1 9.0% by weight 6.0% by weight 1.0% by weight 0.7% by weight 1.0% by weight 1 0 0.0% by weight -17- 201125571 [Example 8] Spraying liquid formulation ethanol citrate trisodium citrate CSD3 water 2 5.0% by weight 1. 5重量% 1.0% by weight 0.5% by weight Balance 1 0 0.0% by weight [Example 9] Chewing gum formula Gummy sugar Glucose starch syrup Perfume CSD3 2 0.0% by weight 5 4.7 wt% 1 5.0 wt% 9.3 wt% 0.5 wt% 0.2 wt% 1 0 0.0 weight % [Example 10] Confectionery Formula Granulated Sugar Syrup Citric Acid Fragrance CSD3 Water 5 0.0% by weight 3 4.0% by weight 1.0% by weight 0.2% by weight 0.4% by weight Balance 1 0 0.0% by weight -18- 201125571 [Example 1 1 ] Ingot formula granulated sugar sucrose fatty acid ester perfume CSD3 water 7.6 wt% 1 9.0 wt% 0.2 wt% 0.2 wt% 0.5 wt% balance 1 0 0.0 wt% [Example 1 2 ] Beverage formula Orange juice isomerization sugar citrate Vitamin C Spice CSD3 Water 3 0.0 0% by weight 1 5.2 4% by weight 0 . 1 0% by weight 〇. 0 4% by weight 0.1 0% by weight 0 . 1 0% by weight 1 0 0.0 0% by weight [Example 13] Powder formulation Corn starch Carboxy cellulose CSD3 5 5.0% by weight 4 0.0% by weight 5.0% by weight 1 0 0.0% by weight [Example I4] Tablet formulation Lactose crystalline cellulose hard Magnesium citrate CSD3 7 0.0% by weight 1 5.0% by weight 5.0% by weight 1 0.0% by weight 1 0 0.0% by weight -19- 201125571 [Example 15] Mouthwash syrup formula Ethanol flavor saccharin chlorhexidine hydrochloride CSD3 water 2.0 0% by weight 1.0 0重量% 0.0 5 wt% 0.0 1 wt% 〇. 5 0 wt% balance 1 0 0.0 0 weight 0 / 〇 [Example 16] Gummy formula gelatin starch syrup sugar plant oil mannitol lemon juice CSD3 6 0.0 0% by weight 2 3.0 0 weight % 8 · 50 0% by weight 4.5 0% by weight 2.9 5% by weight 1. 0 0% by weight 0.0 5% by weight 100.00% by weight [Simplified illustration] [Fig. 1] shows the destructive activity of the chess extract on the viral envelope . Fig. 2 shows inhibition of adsorption activity, VRNA synthesis inhibitory activity, mRNA synthesis inhibitory activity, and cRNA synthesis inhibitory activity of the plant extract. [Fig. 3] shows the hemolysis inhibitory activity of the chess extract. -20-

Claims (1)

201125571 VII. Scope of application for patents: 1. An anti-influenza virus agent with the extract of Chaenomeles sinensis (Pseudocydonia sinensis) as an active ingredient. 2. An anti-influenza virus agent which is obtained by extracting a mold using a 50% aqueous solution of ethanol, and extracting the extract obtained by column fractionation purification as an active ingredient. An influenza virus adsorption inhibitor is obtained by extracting a mold using a 50% aqueous solution of ethanol, and extracting the extract by column fractionation as an active ingredient. A viral mRNA synthesis inhibitor for infecting influenza virus cells, which is obtained by extracting a 50% aqueous solution of ethanol, and extracting the extract by column fractionation as an active ingredient. 5. A virus vRN A synthesis inhibitor that infects influenza virus cells, which is extracted by using a 50% aqueous solution of ethanol, and then the extract obtained by column fractionation purification is effective. ingredient. 6. A virus CRNA synthesis inhibitor for infecting influenza virus cells, which is obtained by extracting a mold using a 50% aqueous solution of ethanol, and using the extract obtained by column fractionation purification as an active ingredient. . 7. A hemolytic inhibitor for a virus membrane fusion activity test, which is obtained by extracting a mold using a 50% aqueous solution of ethanol, and extracting the extract obtained by column fractionation purification as an active ingredient. 8. A film breaker for influenza virus, which is obtained by extracting a mold using a 50% aqueous solution of ethanol, and extracting the extract by column fractionation as an active ingredient. -21 - 201125571 9. A food or drink containing a molded extract and having an anti-epidemic cold virus effect. -twenty two-