JP6507345B2 - Composition for antiviral - Google Patents

Description

The present invention relates to novel antiviral compositions and their uses, and more particularly to antiviral compositions which inactivate viruses at low concentrations and their uses.

  Viruses include viruses that infect humans such as avian influenza virus, norovirus, rotavirus, SARS, coronavirus, retroviruses, pathogenic viruses including viruses that infect crops such as tobacco mosaic virus, and bacteria There are bacterial viruses (also called bacteriophages or phages) that cause lytic action.

  For prevention and removal of pathogenic viruses, generally, disinfection with ethanol, sodium hypochlorite or the like and administration of a vaccine are given. Norovirus is a food-borne virus that causes non-bacterial acute gastroenteritis when it infects humans, so Norovirus control has become one of the important food hygiene measures. Norovirus belongs to a non-membrane virus of RNA type which is difficult to make antiviral drug. Treatment of patients with food poisoning and infections with such viruses provides symptomatic treatment. It is important to inactivate the virus and prevent the virus infection before it is difficult to develop an antiviral drug and to counteract a virus that can only be treated after symptomatic treatment after infection.

  Phage is used to treat bacterial infections against pathogenic multidrug resistant bacteria (phage therapy), obtain approval of food additives by the FDA as a phage preparation for listeria causing food poisoning, and the like. Furthermore, techniques using the phage-derived enzyme endolysin as an antibacterial agent have also been proposed. (Patent documents 1 and 2).

  On the other hand, in the fermentation industry using useful bacteria such as Bacillus natto and lactic acid bacteria, phages infect useful bacteria to cause problems of preventing fermentation and commercialization. Filters and air filters are used to prevent phage contamination, but they are not complete. When phage contamination occurs, it is a common measure to stop production and kill all contaminants including phage with a bactericide before resuming production.

  Antimicrobial agents such as bleached powder (calcium hypochlorite), alkyldimethylbenzyl ammonium chloride, formalin, pyrocarbonic acid ethyl ester, elevation, lead acetate, hydrogen peroxide and the like have been used as preventive measures for phage contamination (non-patent literature) 1). The use of novel anti-phage agents, such as L-ascorbic acid derivatives, has also been proposed (US Pat. No. 5,677,859). However, now that regulations on chemical safety have become stricter, many antimicrobial agents can not be used. Moreover, it is not preferable to use an antibacterial agent or an anti-phage agent at a high concentration in terms of preventing the fermentation of useful bacteria.

  Methods are also taken in which alternative strains and phage insensitive strains are maintained and stored, and when phage contamination occurs, production is switched to alternative strains and insensitive strains (Non-patent Document 1). For phage contamination with temperite phage in lactic acid fermentation, a countermeasure by desolvation strain isolation is also effective (Non-patent Document 1). However, preparation of alternative strains and equipment such as a fermenter are required twice, and the burden is large.

  Single use or mixed use of phage resistant strains has also been proposed. In the preparation of phage-resistant strains, a method of selecting phage-resistant bacteria by causing phages to act directly on bacteria for production by fermentation (Patent Documents 4 and 5), and phage-resistant bacteria by acting phages directly as lysogens There is a method (patent document 6) which avoids the possibility of becoming, and a genetically modified bacterium (patent document 7) to which phage resistance is imparted. However, phage resistant strains affect product quality such as flavor. The practical application of genetic recombination has many problems in terms of regulation and safety evaluation for microorganisms.

Special Table 2013-532955 JP 2013-81465 Tokuhei 4-72802 JP 63-216449 Patent No. 3863958 Patent 3027460 US2006 / 0019370A Patent document 1: JP-A-2005-278549 Patent No. 5084748 JP, 2009-0882064

"Lactic acid bacteria and bifidobacteria science" edited by the Japanese Society of Lactic Acid Bacteria, published by Kyoto University Academic Press, November 2010 Volker Fintermann et al., "Physiotherapy Encyclopedia", 12th Edition, published by Gai Abx Phytochemistry Vol. 58, Issue 3, p. 441-449, 2001

  An object of the present invention is to provide a novel safe and effective antiviral composition in a situation where a technique for inactivating a virus has not been established.

As a result of intensive studies on the above problems, the present inventors have found that antiviral compounds are naturally derived from safe compounds, and have found that they can be applied to subjects requiring antiviral properties, thereby completing the present invention. That is, the present invention relates to Formula (1):
[Wherein, M ₁ and M ₂ are each independently a hydrogen atom, an alkali metal atom or an alkaline earth metal atom]
There is provided an antiviral composition comprising, as an active ingredient, 2H-pyran-2-one-4,6-dicarboxylic acid represented by the formula, an alkali metal salt thereof, or an alkaline earth metal atom thereof. Patent Document 9 discloses the use of 2H-pyran-2-one-4,6-dicarboxylic acid or its alkali metal salt as an antibacterial agent and a bactericidal agent. However, Patent Document 9 is novel in that the antiviral agent is neither described nor suggested. Herein, 2H-pyran-2-one-4,6-dicarboxylic acid, an alkali metal salt thereof or an alkaline earth metal salt thereof is referred to as PDC and the like.

  The virus is, for example, a phage.

The invention also relates to Formula (1):
[Wherein, M ₁ and M ₂ each independently represent a hydrogen atom, an alkali metal atom or an alkaline earth metal atom]
An antiviral composition comprising 2H-pyran-2-one-4,6-dicarboxylic acid represented by the formula, an alkali metal salt thereof or an alkaline earth metal salt thereof as an active ingredient is a subject requiring an antiviral Provided is a method for preventing virus contamination, which comprises adding.

  The use of the antiviral composition may be such that the concentration of PDC or the like is 10 to 300 μg / mL with respect to the volume of the subject requiring the virus (for example, the volume of the fermented substance in the fermenter) preferable.

  The subject in need of the antiviral is, for example, a raw material of fermented food using useful bacteria.

  The virus is, for example, a phage.

  According to the composition for antivirals of this invention which mix | blended PDC etc., a virus can be inactivated safely and simply. The compositions of the present invention are applicable to both non-membrane and filmed viruses. In particular, it has an excellent feature of being applicable to non-membrane virus which has few effective means for inactivating the virus. Taking advantage of this property, the composition of the present invention is expected to impart antiviral properties to pharmaceuticals, industrial products, foods and the like, in addition to the use as a disinfectant for antivirals.

  It is known from Patent Document 9 that PDC or its alkali metal salt has bactericidal activity. Its antibacterial and bactericidal activity is first demonstrated at high concentrations. For example, the minimum growth inhibitory concentration of PDC against bacteria is 2,500 μg / mL in E. coli and 1,250 μg / mL in S. aureus. The minimum bactericidal concentration of B. subtilis is 1,000 μg / mL or more. Therefore, in order to use PDC or its alkali metal salt for antibacterial and sterilizing, a minimum use concentration of 0.1 to 5% is required depending on bacteria. On the other hand, the minimum concentration at which PDC or its alkali metal salt exhibits antiviral activity is exerted at a minimum use concentration as low as about 250 μg / mL. Taking advantage of the fact that the optimum range of the concentration showing antivirality is lower than the antibacterial property, the method for preventing viral contamination of the present invention can be used for applications where it is desired to use useful bacteria to kill only virus. For example, the antiviral composition according to the present invention is directly added at a low concentration to a fermenter containing useful bacteria such as lactic acid bacteria and Bacillus subtilis to prevent phage contamination without affecting useful bacteria contributing to fermentation.

  Hereinafter, an embodiment of the present invention will be described in more detail. The virus targeted by the present invention is not particularly limited. Viruses are roughly divided into two types depending on whether the genome is DNA or RNA, and further classified according to whether they are membraneed viruses whose capsid is covered by an envelope consisting of a lipid bilayer membrane or non-enveloped viruses that are not covered by an envelope. Ru. Specifically, human herpesvirus, hepatitis B virus and the like for DNA type filmed viruses; adenovirus, B19 virus and the like for DNA type filmless viruses; influenza virus, SARS for RNA type filmed viruses Coronaviruses and the like; and RNA-type non-membrane viruses include norovirus, poliovirus, enterovirus and the like. Also in phages which infect bacteria, there are filmed viruses such as φ6 phage and non-membrane viruses such as Qβ phage and MS2 phage.

The antiviral composition of the present invention has the formula (1):
[Wherein, M ₁ and M ₂ each independently represent a hydrogen atom, an alkali metal atom or an alkaline earth metal atom]
And 2H-pyran-2-one-4,6-dicarboxylic acid (hereinafter referred to as PDC), an alkali metal salt thereof or an alkaline earth metal salt thereof as an essential active ingredient.

  Examples of the above alkali metals include lithium, sodium and potassium. Examples of the above-mentioned alkaline earth metals include magnesium, calcium, strontium and barium.

  The manufacturing method of PDC etc. is not specifically limited. For example, it can be obtained by extraction from natural plants, microbial fermentation or chemical synthesis. Natural plants containing the PDC (scientific name Geum urbanum: English name Wood avens) have been used for a long time in phytotherapy (Non-patent documents 2 and 3). From these plants, PDC can be directly extracted by a conventional method.

  For example, according to the production method described in Patent Document 10, PDC is a transformed cell in which a fermentation production plasmid pCDFDuet-qutC and pULABC has been introduced into Escherichia coli XL1-Blue strain (STRATEGENE, CA, USA) using saccharides as a starting material according to the production method described in By culturing the Alternatively, according to the microbial fermentation method described in Patent Document 8, the fermentation production plasmid pKTVLABC of PDC was introduced into Pseudomonas bacteria (Pseudomonas putida PbY1100) using vanillin, syringaaldehyde, vanillic acid, syringic acid or protocatechuic acid as a starting material. Transformed cells may be cultured. PDC may be extracted and purified from the culture according to a standard method. The contents described in Patent Documents 8 and 10 are herein incorporated by reference.

  PDC etc show high water solubility. The antiviral composition of the present invention can be easily prepared by dissolving PDC or the like at an arbitrary concentration in an aqueous medium such as water.

  The concentration of the formula (1) in the antiviral composition may generally be 0.001 to 0.09 wt%, preferably 0.002 to 0.05 wt%, particularly preferably 0.002 It is 0.03% by weight.

  In addition to the active ingredient of the formula (1), the antiviral composition of the present invention can be used as an auxiliary agent of an antiviral agent without particular limitation, without departing from the scope of the effects of the present invention. Examples of such auxiliary agents include known antiviral agents, chelating agents, pH adjusters, surfactants, ultraviolet light absorbers, antioxidants, emulsifiers, perfumes, coloring agents, water-soluble polymers, alcohols, A fluorescent whitening agent, a viscosity modifier, a foaming agent etc. are mentioned.

  The form of the composition of the present invention is not particularly limited, and includes aqueous solutions, sprays, creams, pastes, gels, gels and the like. Since the composition of the present invention is water soluble, it is particularly advantageous to use it as an aqueous solution, an antiviral agent in the form of a spray.

The invention also relates to Formula (1):
[Wherein, M ₁ and M ₂ each independently represent a hydrogen atom, an alkali metal atom or an alkaline earth metal atom]
An antiviral composition comprising 2H-pyran-2-one-4,6-dicarboxylic acid represented by the formula, an alkali metal salt thereof or an alkaline earth metal salt thereof as an active ingredient is a subject requiring an antiviral Provided is a method for preventing virus contamination, which comprises adding. The usage of the method for preventing virus contamination is exemplified below.

  Subjects that require the aforementioned antivirals include pharmaceuticals, agricultural chemicals, medicines such as disinfectants, and quasi-drugs; air cleaning related products, deodorants, cosmetics, detergents, resin moldings, coatings, Industrial products such as adhesives; Food, feed, etc. are included.

  Anti-viral disinfectant disinfects fingers etc. while packed in a manual or automatic dispenser, and is impregnated in a carrier such as paper, non-woven fabric, cloth, sponge etc. (wet wiper etc.) toilet seat, kitchen, table, floor Etc. are used to disinfect the substrate. The antiviral disinfectant is produced by dissolving or dispersing the composition of the present invention in an aqueous solvent such as water, alcohol and the like and, if necessary, adding known adjuvants with the antiviral disinfectant.

  The deodorant comprising the composition of the present invention imparts antiviral properties to the deodorant. This deodorant is produced by dissolving or dispersing the composition of the present invention in an aqueous solvent, and optionally adding an auxiliary agent such as a fragrance and a masking agent. The antiviral deodorant is, for example, packed in a spray and then sprayed on indoor space (living room, toilet, etc.), clothes, bedding, curtains, furniture, and the like.

  The composition of the present invention can also be used by kneading it into a resin molding made of polyurethane or melamine resin. After the composition of the present invention is blended with a stock solution in which a polyol, a foaming agent, a foaming agent, a catalyst and the like are mixed in advance, it is foamed and cured by mixing with an isocyanate compound to produce a polyurethane foam. A part of the polyol component and the composition of the present invention may be premixed to prepare a masterbatch, which may be mixed with other components just before foaming to produce a foam. In addition, the composition of the present invention is dissolved or dispersed in a melamine resin solution before curing, and impregnated in decorative paper or the like, and then heat cured to form a decorative board. A filler can be added to the melamine resin solution before curing, and pressure and heat curing can be performed to form a container or the like. Foams are used, for example, as mattresses, bedding such as pillows, foam cleaners, etc., and melamine resins are used as furniture and decorative boards.

  When the composition of the present invention is added to air cleaning related products such as a mask, an air cleaner, an air conditioner, etc. to impart antiviral properties, air such as a mask, filter, scrubber liquid, etc. made of non-woven fabric or foam. By impregnating, dropping, spraying or adding an aqueous solution or aqueous dispersion of the composition of the present invention to a cleaning member, or by kneading the composition of the present invention into fibers or foam itself that is the basis of a mask or filter, Make an antiviral mask, an antiviral filter and an antiviral scrubber liquid.

  The composition of the present invention may be added to a coating agent such as a paint or ink, or may be kneaded into a resin molding to impart antiviral properties. The places where they are used include, for example, interior materials such as residences, offices, automobiles, railway cars, aircraft walls, floors, and ceilings; and fixtures such as tables, table cloths, and kitchen stands. Moreover, when adding to coating agents, such as a coating material and an ink, it is good to add to aqueous dispersions, such as water-soluble resin like a melamine resin, or acrylic resin, acrylic silicone resin, a polyurethane resin, for example.

  In the usage mode for imparting antiviral properties to the above various products, the lower limit of the concentration of the PDC or the like relative to the volume of the subject requiring antiviral may be usually 10 μg / mL, preferably 25 μg / mL, more preferably It is 30 μg / mL. Although the upper limit is not particularly limited, it is usually 500 μg / mL, preferably 300 μg / mL.

  The method for preventing viral contamination of the present invention is preferably carried out to prevent viral contamination, particularly phage contamination, during the production of a fermented food using useful bacteria. Specific examples of useful bacteria / fermented foods include yeast / wine, beer, sake, etc .; lactic acid bacteria / yoghurt; yeast / breads; Bacillus subtilis (especially, natto bacteria) / natto; bacillus / Japanese sake, miso, sweet sake, etc .; Acetic acid bacteria / vinegar is included. At the time of production of a fermented food using useful bacteria, the antiviral agent composition of the present invention is added to a fermentation raw material so that the PDC concentration falls within the following range.

  In the usage mode of inactivating the virus while utilizing the above-mentioned useful bacteria, the lower limit of the concentration such as PDC with respect to the volume of the subject requiring antiviral (for example, the volume of fermented matter in the fermenter) is usually 10 μg / ML, preferably 25 μg / mL, particularly preferably 50 μg / mL. The upper limit is usually 1,000 μg / mL, preferably 500 μg / mL, particularly preferably 300 μg / mL. If the upper limit exceeds 1,000 μg / mL, the useful bacteria may be killed depending on the type of useful bacteria.

  Hereinafter, the present invention will be described in more detail by way of examples of the present invention. However, the present invention is not limited to the examples.

Example 1 Antiviral Test on Membraneless Phage 1. Preparation of composition for antivirals By diluting PDC synthesized and purified according to the microbial fermentation method described in Patent Document 10 in water, PDC concentration 10 μg / mL, 25 μg / mL, 50 μg / mL, 250 μg / mL, 500 μg / mL. Compositions of mL and 2500 μg / mL were prepared. In addition, the composition of 500 μg / mL of PDC (pH 3.0 / Na adjustment) was adjusted to pH 3.0 with an aqueous NaOH solution at the time of preparation.

2. Antiviral test 10 μL of phage solution of Qβ phage (NBRC 20012) (6.5 × 10 ⁹ PFU / mL: Table 1, 3.5 × 10 ¹⁰ PFU / mL: Table 2), water (blank) or the present invention It was added to 1 mL of the composition and allowed to stand for the time shown in Tables 1 and 2.
Thereafter, a neutralizing agent was immediately added to stop the action of the composition of the present invention on the virus.

  The infective titer of the phage was measured by the plaque counting method using the solution in which the action was stopped as a sample stock solution for infective titer measurement. Specifically, a host bacterium E. coli obtained by culturing a stock solution stepwise diluted with PBS (phosphate buffered saline) in the form of a sheet was used. After contacting with Coli (NBRC 106373), the whole was covered with agar and cultured so as not to spread the virus. Since the cells infected with the virus change in shape (cytopathic), the infected cells (plaques) were counted to quantify the infectivity titer of the virus.

Furthermore, the infective titer log reduction value was determined by inserting the infectivity titers of blanks and samples with Qβ phage into the following formula.

[Example 2] Antiviral test on filmed phage In Example 1, 10 μL of phage solution of φ 6 phage (NBRC 105899) (3.9 × 10 ⁷ PFU / mL: Table 3, 3.9 × 10 ⁸ PFU) / ML: changed to Table 4), and the host strain was P. The antiviral test was performed in the same manner as in Example 1 except that it was changed to Syringae (NBRC 105640). The measurement results of the infectivity titer and the infective titer log reduction value of the φ6 phage are shown in Tables 3 and 4.

Example 3 Antiviral Test Against Influenza A Virus (H3N2) Preparation of Composition for Antiviral Composition The PDC synthesized and purified in Example 1 was diluted in water to prepare a composition with a PDC concentration of 250 μg / mL and 25 μg / mL.

2. Antiviral test 1 mL (4.5 × 10 ⁷ PFU / mL) of a test virus suspension of influenza A virus (H3N2) is added to 9 mL PBS (control) or the composition of the present invention for 5 minutes at 25 ° C. , Left. After standing, 0.5 mL of the mixture was added to and mixed with 4.5 mL of a drug inactivating agent (SCDLP medium). The virus infectivity titer of these mixed solutions to MDCK cells (dog kidney-derived cells), which are host cells, was measured by the plaque measurement method in the same manner as in Example 1. Table 5 shows the results.

  The antiviral composition of the present invention was confirmed to exhibit antiviral properties at 250 μg / mL or less, that is, at a concentration lower than the concentration at which the antibacterial effect against various fungi is exhibited. This has made it possible to provide an effective and convenient antiviral agent against phage contamination in the fermentation industry.

  Inactivation of membraneless viruses and phages by conventional antiseptics such as ethanol is considered to be less effective than filmed viruses and phages. From the comparison of Examples 1 and 2, it was found that inactivation of a membraneless virus or phage by the composition of the present invention exerts effects more than a filmed virus or phage.

  Inactivation of membraneless viruses and phages by conventional antiseptics such as ethanol is considered to be less effective than filmed viruses and phages. From the comparison of Examples 1 and 2, it was found that inactivation of a membraneless virus or phage by the composition of the present invention exerts effects more than a filmed virus or phage.

Claims (5)

Formula (1):
[Wherein, M ₁ and M ₂ are each independently a hydrogen atom, an alkali metal atom or an alkaline earth metal atom]
An antiviral composition comprising, as an active ingredient, 2H-pyran-2-one-4,6-dicarboxylic acid represented by or an alkali metal salt thereof or an alkaline earth metal salt thereof.   The composition for antivirals according to claim 1, wherein the virus is a phage. Formula (1):
[Wherein, M ₁ and M ₂ each independently represent a hydrogen atom, an alkali metal atom or an alkaline earth metal atom]
An antiviral composition comprising 2H-pyran-2-one-4,6-dicarboxylic acid represented by the formula, an alkali metal salt thereof or an alkaline earth metal salt thereof as an active ingredient is a subject requiring an antiviral A method for preventing virus contamination, comprising adding
The method for preventing virus contamination, wherein a subject in need of the antiviral is a raw material of a fermented food using useful bacteria .   The concentration of the 2H-pyran-2-one-4,6-dicarboxylic acid, its alkali metal salt or its alkaline earth metal salt is 10 to 1000 μg / mL with respect to the volume of the subject requiring the antiviral The method for preventing viral contamination according to claim 3, characterized in that an antiviral composition is used.   The method for preventing viral contamination according to claim 4, wherein the virus is a phage.