Inorganic antiviral agents

Abstract

To provide an inorganic antiviral agent that exhibits excellent antiviral action on non-enveloped virus as well as enveloped virus.SOLUTION: An inorganic antiviral agent is composed of silica doped with a metal. Examples of the metal include copper, aluminum, zirconium, cobalt, manganese, iron. The inorganic antiviral agent may be used in the manner of being included in the slurry prepared by suspending the metal-doped silica in a dispersion medium.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to inorganic antiviral agents. [Background technology]

[0002] It is well known that in recent years, interest in antiviral agents to combat viral infections has grown due to the emergence of new strains of influenza viruses and coronaviruses that cause severe symptoms upon infection. Various active ingredients for antiviral agents have been proposed to date, and it has long been known that ions of metals such as copper and silver, which are inorganic components with superior chemical stability compared to organic components, exhibit antiviral activity (e.g., Patent Document 1). Furthermore, Patent Document 2 reports that mesoporous silica, a type of porous silica, exhibits antiviral activity against influenza viruses.

[0003] Viruses are broadly classified based on their structure into those with an envelope made of lipids and glycoproteins (enveloped viruses) and those without (non-enveloped viruses). Influenza viruses are a typical example of enveloped viruses, and noroviruses are a typical example of non-enveloped viruses. Non-enveloped viruses are said to be more resistant to alcohol and heat and to have stronger infectivity than enveloped viruses. Therefore, there is a demand for inorganic antiviral agents that exhibit excellent antiviral activity not only against enveloped viruses but also against non-enveloped viruses. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2011-042615 [Patent Document 2] Japanese Patent Application Publication No. 2019-151592 Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, an object of the present invention is to provide an inorganic antiviral agent that exhibits excellent antiviral activity not only against enveloped viruses but also against non-enveloped viruses. [Means for solving the problem]

[0006] As a result of extensive research conducted in light of the above points, the present inventors have found that metal-doped silica exhibits excellent antiviral activity not only against enveloped viruses but also against non-enveloped viruses.

[0007] The inorganic antiviral agent of the present invention, which was developed based on the above findings, comprises aluminum-doped silica, as described in claim 1. (In the above, "aluminum-doped silica" means silica in which aluminum is chemically bonded and incorporated into the inorganic network of siloxane bonds that constitutes silica.) . The inorganic antiviral agent according to claim 2 is the inorganic antiviral agent according to claim 1, further comprising silica doped with at least one metal selected from the group consisting of copper, zirconium, cobalt, manganese, and iron. (In the above, "metal doped into silica" means that the metal is incorporated by chemical bonding into the inorganic network consisting of siloxane bonds that constitute silica.) . The inorganic antiviral agent according to claim 3 is the inorganic antiviral agent according to claim 2, in which copper and aluminum are doped into silica. The inorganic antiviral agent according to claim 4 is the inorganic antiviral agent according to any one of claims 1 to 3, wherein the silica is porous silica. The inorganic antiviral agent according to claim 5 is the inorganic antiviral agent according to any one of claims 1 to 3, wherein the silica is non-porous silica. Furthermore, as set forth in claim 6, the article of the present invention comprises the inorganic antiviral agent set forth in any one of claims 1 to 5. [Effects of the Invention]

[0008] According to the present invention, it is possible to provide an inorganic antiviral agent that exhibits excellent antiviral activity not only against enveloped viruses but also against non-enveloped viruses. DETAILED DESCRIPTION OF THE INVENTION

[0009] The inorganic antiviral agent of the present invention comprises metal-doped silica. Here, "metal-doped silica" refers to silica in which a metal is incorporated by chemical bonding into an inorganic network made up of siloxane bonds that constitutes the silica. The metal-doped silica may be porous or non-porous, and there are no particular limitations on its shape.

[0010] Examples of metals that can be doped into silica include copper, aluminum, zirconium, cobalt, manganese, and iron, which can be used alone or in combination of two or more.

[0011] The metal content in the metal-doped silica (when two or more metals are used in combination, the total amount of each metal) is, for example, 0.01 to 10 wt%, preferably 0.1 to 5 wt%. If the metal content in the metal-doped silica is less than 0.01 wt%, sufficient antiviral activity may not be obtained, while silica doped with a metal in an amount exceeding 10 wt% may be difficult to produce. When two or more metals are used in combination, the ratio of the metal contents may be, for example, 0.1 to 2 times the content of one metal relative to the content of the other metal.

[0012] When the metal-doped silica is porous, the metal-doped porous silica may be, for example, that described by the present inventors in JP-A-2020-15640. Specifically, it is as follows.

[0013] An example of porous silica is mesoporous silica in which pores (mesopores) having a diameter of 2 to 50 nm are regularly arranged.

[0014] The specific surface area of ​​porous silica is, for example, 500 to 2000 m 2 / g is preferable in terms of maintaining durability.

[0015] Metal-doped mesoporous silica can be produced according to the following method, which is known per se, for example, as described in JP-A-2020-15640.

[0016] (Process 1) First, a surfactant and raw materials for doping a metal into mesoporous silica are dissolved in a solvent and stirred, for example, at 30 to 200° C. for 0.5 to 10 hours to form micelles in the surfactant.

[0017] The amount of surfactant dissolved in the solvent is, for example, 10 to 400 mmol / L, preferably 50 to 150 mmol / L, or, for example, 0.01 to 5.0 mol, preferably 0.05 to 1.0 mol, relative to 1 mol of the silica raw material added in Step 2 described below.

[0018] The surfactant may be a cationic surfactant, an anionic surfactant, or a nonionic surfactant, but is preferably a cationic surfactant such as an alkylammonium salt. The alkylammonium salt preferably has an alkyl group containing 8 or more carbon atoms, and from the perspective of industrial availability, an alkyl group containing 12 to 18 carbon atoms is more preferred. Specific examples of alkylammonium salts include hexadecyltrimethylammonium chloride, cetyltrimethylammonium bromide, stearyltrimethylammonium bromide, cetyltrimethylammonium chloride, stearyltrimethylammonium chloride, dodecyltrimethylammonium bromide, octadecyltrimethylammonium bromide, dodecyltrimethylammonium chloride, octadecyltrimethylammonium chloride, didodecyldimethylammonium bromide, ditetradecyldimethylammonium bromide, didodecyldimethylammonium chloride, and ditetradecyldimethylammonium chloride. The surfactant may be used alone or in combination of two or more.

[0019] The amount of raw material to be dissolved in the solvent for doping the metal into the mesoporous silica (the total amount of each raw material when two or more metals are used in combination) is, for example, 0.001 to 0.5 mol, preferably 0.01 to 0.1 mol, per 1 mol of the silica raw material added in Step 2 described below.

[0020] As raw materials for doping mesoporous silica with metals, for example, metal nitrates, sulfates, chlorides, and oxychlorides can be used. When doping with copper, it is preferable to use copper nitrate or copper chloride. When doping with aluminum, it is preferable to use aluminum chloride. When doping with zirconium, it is preferable to use zirconium oxychloride. When doping with cobalt, it is preferable to use cobalt nitrate. When doping with manganese, it is preferable to use manganese chloride. When doping with iron, it is preferable to use iron chloride. The raw materials for doping with metals may be used alone or in combination of two or more.

[0021] The solvent may be, for example, water, or a mixed solvent of water and a water-soluble organic solvent such as methanol, ethanol, diethylene glycol, or glycerin.

[0022] (Process 2) Next, the silica raw material is dissolved in the surfactant-forming micelle solution obtained in step 1, for example, at room temperature, and stirred until homogenous, allowing the silica raw material to accumulate on the surface of the surfactant micelles. The amount of silica raw material dissolved in the solution is, for example, 0.2 to 1.8 mol / L. Alternatively, when water or a mixed solvent of water and a water-soluble organic solvent is used as the solvent, the amount is, for example, 0.001 to 0.05 mol per 1 mol of water.

[0023] The silica raw material is not particularly limited as long as it forms an inorganic network consisting of siloxane bonds that constitute mesoporous silica by dehydration condensation. Specific examples of silica raw materials include tetraalkoxysilanes such as tetraethoxysilane, tetramethoxysilane, and tetra-n-butoxysilane, and sodium silicate. Tetraalkoxysilane is preferred, and tetraethoxysilane is more preferred. The silica raw materials may be used alone or in combination of two or more.

[0024] (Step 3) Next, the silica raw material accumulated on the surface of the surfactant micelles is dehydrated and condensed to form an inorganic network consisting of siloxane bonds that constitute mesoporous silica, and a metal is incorporated into the inorganic network by chemical bonding. The dehydration and condensation of the silica raw material can be carried out, for example, by adding a basic aqueous solution to the system to raise the pH, followed by stirring at room temperature for at least one hour. The basic aqueous solution is preferably added so that the pH is 8 to 14 immediately after addition, and more preferably 9 to 11. Specific examples of basic aqueous solutions include aqueous sodium hydroxide, aqueous sodium carbonate, and aqueous ammonia, with aqueous sodium hydroxide being preferred. The basic aqueous solutions may be used alone or in combination of two or more. The dehydration and condensation of the silica raw material can also be carried out by adding an acidic aqueous solution such as an aqueous hydrochloric acid solution to the system to lower the pH, followed by stirring.

[0025] (Step 4) Finally, the surfactant micelles obtained in step 3, which form an inorganic network on the surface composed of siloxane bonds that constitute the mesoporous silica and to which metals are chemically bonded, are collected as a precipitate by filtration, dried at 30 to 70°C for 10 to 48 hours, for example, and then calcined at 400 to 600°C for 1 to 10 hours to obtain the desired metal-doped mesoporous silica. The metal-doped mesoporous silica obtained in this way may be pulverized in a mixer or mill, if necessary, to obtain the desired particle size (e.g., a median diameter of 0.01 to 100 μm).

[0026] The addition of raw materials to the system for doping metal into mesoporous silica is not limited to the above-mentioned step 1 in which the raw materials are dissolved in a solvent together with a surfactant, but may be dissolved in a solution in step 2 or 3, as long as the silica raw materials are dissolved in the solution until the formation of an inorganic network consisting of siloxane bonds that constitutes mesoporous silica by dehydration condensation in step 3 is completed.

[0027] When the metal-doped silica is nonporous, it can be produced, for example, by a precipitation method, which is a well-known method for producing nonporous silica. Specifically, it can be produced by neutralizing an alkali metal silicate as a silica raw material with a mineral acid while allowing a raw material for doping the metal into the nonporous silica to coexist in the system. The raw material for doping the metal into the nonporous silica may be the same as the raw material for doping the metal into the mesoporous silica. The amount of raw material for doping the metal into the nonporous silica (the total amount of raw materials when two or more metals are used in combination) used is, for example, 0.001 to 0.5 mol, preferably 0.01 to 0.1 mol, per mol of the alkali metal silicate as the silica raw material. The alkali metal silicate used as the silica raw material may be a common one used to produce nonporous silica by a precipitation method. Specific examples include sodium silicate and potassium silicate, and the molar ratio of silica to alkali: SiO2 / MO (where M is Na, K, or the like) is, for example, 0.5 to 4. The amount of alkali metal silicate dissolved in a solvent (such as water) is, for example, 0.001 to 0.05 mol per 1 mol of the solvent. Hydrochloric acid, sulfuric acid, or nitric acid can be used as the mineral acid. The metal-doped nonporous silica is obtained as a precipitate produced by the neutralization reaction of the alkali metal silicate with the mineral acid, and its filtration, washing, drying, etc. may be performed in accordance with the methods used to produce nonporous silica by precipitation. Finally, if necessary, the silica may be calcined at a high temperature of 300°C or higher. The specific surface area (BET specific surface area) of the metal-doped nonporous silica thus produced is, for example, 0.1 to 350 m2 / g. If necessary, it may be pulverized in a mixer or mill to have a desired particle size (for example, a median diameter of 0.01 to 100 μm), just like the above-mentioned metal-doped mesoporous silica.

[0028] The inorganic antiviral agent of the present invention can be used as a material for imparting antiviral properties to everyday items such as textile products, nonwoven fabric products, leather products, building materials, wood, paints, adhesives, plastics, films, ceramics, paper, pulp, metal processing oils, water treatment agents, cosmetics, stationery, toys, containers, caps, spouts, and other items. The manner of use may be the same as that of conventionally known inorganic antiviral agents. For example, by incorporating the agent into a paint or the like and applying it to the surface of an item, or by incorporating the agent into or supporting it on a material for manufacturing an item (such as paper, fiber, nonwoven fabric, or resin) to manufacture the item, the agent can be used to impart antiviral properties to the item. Furthermore, when the item is a container or the like, the agent can also be expected to exert an antiviral effect on the contents enclosed or filled in the item. Furthermore, the inorganic antiviral agent of the present invention can be formed into pellets or the like and mixed with the item, or used alone.

[0029] The inorganic antiviral agent of the present invention may also be used in a form in which metal-doped silica is contained in a slurry prepared by suspending the metal-doped silica in a dispersion medium. By adopting such a form, the inorganic antiviral agent of the present invention can be incorporated with excellent dispersibility into various liquid products such as detergents, sprays, and aerosols. The metal-doped silica may be suspended in a dispersion medium so that the content of the metal-doped silica in the slurry is, for example, 0.1 to 30 wt%, preferably 0.5 to 20 wt%, and more preferably 1 to 15 wt%. Suspending metal-doped silica in a dispersion medium in an amount such that the content of the metal-doped silica in the slurry exceeds 30 wt% may be difficult due to increased viscosity. Meanwhile, a slurry with a metal-doped silica content of less than 0.1 wt% may result in the metal-doped silica not fully exerting its antiviral effect. For example, water may be used as the dispersion medium. The water used as the dispersion medium may contain a water-soluble organic solvent, such as methanol, ethanol, or a polyhydric alcohol such as diethylene glycol or glycerin, but the water content is preferably 50 wt % or more. The pH of the dispersion medium is, for example, 5 to 11, preferably 6 to 9. If the pH of the dispersion medium is below 5, the metal doped in the silica may dissolve, while if the pH of the dispersion medium is above 11, the silica may dissolve.

[0030] The inorganic antiviral agent of the present invention exhibits an excellent antiviral effect not only against enveloped viruses but also against non-enveloped viruses. Therefore, the type of virus that can be the target of its antiviral effect is not limited, and various pathogenic viruses, for example, enveloped viruses include influenza A, B, and C viruses, parainfluenza virus, mumps virus, measles virus, human metapneumovirus, respiratory syncytial virus, Nipah virus, Hendra virus, yellow fever virus, dengue virus, Japanese encephalitis virus, West Nile virus, hepatitis B, C, and D viruses, eastern and western equine encephalitis virus, rubella virus, Lassa virus, Junin virus, Machupo virus, Ganarito virus, Sabia virus, Crimean-Congo hemorrhagic fever virus, sandfly fever virus, hantavirus, Sin Nombre virus, rabies virus, Ebola virus, Marburg virus, bat leukemia virus, and the like, can be used. Examples of viruses that can be targeted include Sarsaf virus, human T-cell leukemia virus, human immunodeficiency virus, human coronavirus, SARS coronavirus, SARS-CoV-2 virus (COVID-19 virus), herpes virus, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus, smallpox virus, monkeypox virus, cowpox virus, molluscipox virus, parapox virus, and Onyongnyong virus. Examples of non-enveloped viruses that can be targeted include rhinovirus, poliovirus, foot-and-mouth disease virus, rotavirus, norovirus, enterovirus, hepatovirus, astrovirus, sapovirus, hepatitis A virus, hepatitis E virus, human parvovirus, polyomavirus, human papillomavirus, adenovirus, and coxsackievirus. [Example]

[0031] The present invention will be described in detail below with reference to examples, but the present invention should not be construed as being limited to the following description.

[0032] Preparation Example 1: Preparation of copper and aluminum doped mesoporous silica Hexadecyltrimethylammonium chloride (surfactant), copper chloride (copper chloride), and aluminum chloride (aluminum chloride) were dissolved in water and stirred at 100°C for 1 hour. The mixture was then cooled to room temperature and tetraethoxysilane (silica precursor) was further dissolved and stirred until homogeneous. A basic aqueous solution of sodium hydroxide was then added to the reaction mixture, adjusting the pH to 9 immediately after addition, and the mixture was stirred at room temperature for 20 hours. The resulting precipitate was collected by filtration, dried at 50°C for 24 hours, and then calcined at 570°C for 5 hours to obtain the desired copper- and aluminum-doped mesoporous silica as a slightly bluish white powder.

[0033] The amounts of hexadecyltrimethylammonium chloride as a surfactant, copper chloride as a raw material for doping copper into mesoporous silica, aluminum chloride as a raw material for doping aluminum into mesoporous silica, and water as a solvent were as follows, relative to 1 mol of tetraethoxysilane as a silica raw material: Hexadecyltrimethylammonium chloride: 0.225 mol Copper chloride: 0.0204 mol Aluminum chloride: 0.0482 mol Water: 125mol To prepare a sodium hydroxide aqueous solution as a basic aqueous solution, 0.195 mol of sodium hydroxide was used per 1 mol of tetraethoxysilane as a silica raw material.

[0034] The copper and aluminum doped mesoporous silica obtained by the above method has a specific surface area of ​​1100 m 2The adsorption isotherm of nitrogen gas was measured at liquid nitrogen temperature using a BELSORP MAX II model manufactured by MicrotrackBell using the multipoint method, and calculated using the BJH calculation. The same applies below.) Approximately 50 mg of the copper- and aluminum-doped mesoporous silica was accurately weighed and dissolved in 4 mL of hydrochloric acid. The concentrations of copper and aluminum in the hydrochloric acid solution were measured using an inductively coupled plasma optical emission spectrometer (ICP-OES manufactured by Thermo Scientific; the same applies below). Based on the measurement results, the copper and aluminum contents in the copper- and aluminum-doped mesoporous silica were calculated to be 1.78 wt% and 1.84 wt%, respectively. The doping of the mesoporous silica with copper and aluminum was confirmed using an X-ray photoelectron spectrometer (K-Alpha Surface Analysis manufactured by Thermo Scientific, the same applies hereinafter) and a transmission electron microscope (JEM2010 manufactured by JEOL, the same applies hereinafter).

[0035] Preparation Example 2: Preparation of aluminum-doped mesoporous silica Aluminum-doped mesoporous silica was obtained as a white powder (specific surface area: 1151 m) in the same manner as in Production Example 1, except that copper chloride, which was used in Production Example 1 as a raw material for doping copper into mesoporous silica, was not used. 2 / g, pore diameter: 2.5 nm, aluminum content: 1.82 wt%).

[0036] Preparation Example 3: Preparation of a slurry in which copper and aluminum doped mesoporous silica is suspended in a dispersion medium The copper- and aluminum-doped mesoporous silica produced in Production Example 1 was pulverized in a mixer to a median diameter of approximately 25 to 31 μm, and 11 g of the pulverized silica was then placed in a 250 mL polypropylene pot (Eye-Boy PP wide-mouth bottle: AS ONE Corporation; the same applies hereinafter) with 99 g of ion-exchanged water as a dispersion medium and 2 mm diameter alumina balls (Nikkato Corporation; alumina purity: 93%, bulk density: 3.6 g / cm) as a medium. 3, the same below) was placed in the slurry, and wet-pulverized in a pot mill for 8 hours. The alumina balls were then filtered off to obtain a slurry (pH: approximately 7) containing 10 wt% copper- and aluminum-doped mesoporous silica, with a median diameter of 0.50 μm. The X-ray diffraction intensity of the mesoporous silica was measured after filtering and drying the fine particles in the slurry, and the d-value peaked at 3.8 nm (the median diameter was measured using a laser diffraction particle size distribution analyzer (SALD-3100, manufactured by Shimadzu Corporation); the same below).

[0037] Production Example 4: Production of a slurry in which aluminum-doped mesoporous silica is suspended in a dispersion medium A slurry (pH: approximately 7) containing 10 wt % aluminum-doped mesoporous silica with a median diameter of 0.55 μm was obtained in the same manner as in Production Example 3, except that the aluminum-doped mesoporous silica produced in Production Example 2 was used instead of the copper and aluminum-doped mesoporous silica produced in Production Example 1.

[0038] Test Example 1: Antiviral effect of metal-doped mesoporous silica (1) Antiviral effect against enveloped viruses MDCK cells (canine kidney-derived cells) were infected with influenza A virus (H3N2, A / Hong Kong / 8 / 68; TC adapted, ATCC VR-1679) as host cells, and after incubation, the cells were centrifuged to remove cell debris. The virus suspension (infectious titer: 5.2 × 10 8A viral titer of 1.0 PFU / mL was obtained. 1.0 mL of this virus suspension was added to 9.0 mL of a 10 wt% copper- and aluminum-doped mesoporous silica slurry prepared in Preparation Example 3, which was dispersed in ion-exchanged water, and the mixture was left at 25°C for 2 hours. After the suspension, 0.5 mL of the test solution was mixed with 4.5 mL of SCDLP medium as a drug deactivator. 0.1 mL of the resulting mixture was added to 0.9 mL of EMEM medium to terminate the drug reaction, and the viral infectivity of the test solution was measured using the plaque assay. The results are shown in Table 1. Table 1 also shows the results for the use of 9.0 mL of a 10 wt% aluminum-doped mesoporous silica slurry prepared in Preparation Example 4, which was dispersed in ion-exchanged water, and the results for the use of 9.0 mL of phosphate-buffered saline (PBS) as a control.

[0039] (2) Antiviral activity against non-enveloped viruses CRFK cells (cat kidney-derived cells) were used as host cells, and feline calicivirus (F-9, Feline calicivirus Strain: F-9, ATCC VR-782) and cultured. After centrifugation to remove cell debris, the virus suspension (infectivity titer: 2.1 × 10 8 A viral titer of 1.0 PFU / mL was obtained. 1.0 mL of this virus suspension was added to 9.0 mL of a 10 wt% copper- and aluminum-doped mesoporous silica slurry prepared in Preparation Example 3, which was used as a sample, in ion-exchanged water. The mixture was then left at 25°C for 2 hours. After the incubation, 0.5 mL of the test solution was mixed with 4.5 mL of SCDLP medium supplemented with fetal bovine serum (FBS) as a drug inactivator to a final concentration of 10%. 0.1 mL of the resulting mixture was added to 0.9 mL of EMEM medium to terminate the drug reaction. The virus infectivity of the test solution was then measured using the plaque assay. The results are shown in Table 1. Table 1 also shows the results for the use of 9.0 mL of a 10 wt% aluminum-doped mesoporous silica slurry prepared in Preparation Example 4, which was used as a sample, in ion-exchanged water, and the results for the use of 9.0 mL of phosphate-buffered saline as a control.

[0040] (3) Protein adsorption and proteolysis The protein adsorption and proteolytic actions, which are thought to be the mechanisms behind the antiviral effect (Development, Evaluation and Processing Technology of Antibacterial and Antiviral Materials, edited by the Technical Information Association, 2013, pp. 19-26), were evaluated as follows. (a) Protein adsorption 5 mg of the copper- and aluminum-doped mesoporous silica prepared in Preparation Example 1 was added to 0.8 mL of a 1 mg / mL BSA solution prepared by diluting bovine serum albumin (BSA) reagent (Thermo Fisher Scientific, product number 23210) with ion-exchange water. The mixture was stirred for 30 seconds using a vortex mixer and then allowed to stand. After 2 hours, the BSA concentration in the supernatant was measured by colorimetric assay using a Thermo Fisher Scientific Pierce 660 nm Protein Assay kit. The amount of BSA adsorbed per mg of sample was calculated by the amount of BSA reduced from the amount of BSA used in the test. The results are shown in Table 2. Table 2 also shows the results for 5 mg of the aluminum-doped mesoporous silica prepared in Preparation Example 2. (a) Protein decomposition A 1.5 mL polypropylene microtest tube was charged with 200 μg of BSA (Wako Pure Chemical Industries, Ltd., globulin-free) and 250 mg of the copper- and aluminum-doped mesoporous silica slurry prepared in Preparation Example 3 as a sample. The total volume was adjusted to 1 mL with ion-exchanged water and the tube was shaken at room temperature on a shaker to allow contact between the BSA and the sample. After 60 hours, 0.8 mL of the supernatant was collected by centrifugation. 0.64 mL of 10% SDS solution and 0.16 mL of ion-exchanged water were added to the remaining 0.2 mL, and the tube was shaken at room temperature on a shaker to separate the BSA and its degradation products electrically adsorbed on the surface of the copper- and aluminum-doped mesoporous silica contained in the sample. After 2 hours, the BSA and its degradation products contained in the 0.8 mL of supernatant collected by centrifugation were visually observed by polyacrylamide gel electrophoresis. The protein concentration of the supernatant recovered by centrifugation was quantified using BCA reagent and diluted with ion-exchanged water to the same concentration as the blank BSA solution (40 μg / mL). 21.84 μL of the resulting solution was mixed with 2.18 μL of 1M dithiothreitol, 19.65 μL of 2× SDS PAGE Sample Buffer (TCI), and 1.31 μL of 1M Tris-HCl buffer (Fujifilm Wako Pure Chemical Industries, Ltd.) in a 1.5 mL polypropylene microtest tube, heated to 95°C for 7 minutes, and loaded onto a polyacrylamide gel. The polyacrylamide gel used was a 1.0 mm, 12+2 well comb Criterion TGX Precast Gels (BIO RAD). After electrophoresis at 200 V, the gel was stained with BCC reagent and proteolysis was assessed based on band morphology.

[0041] [Table 1]

[0042] [Table 2]

[0043] As is clear from Table 1, both the slurry containing copper- and aluminum-doped mesoporous silica produced in Production Example 3 and the slurry containing aluminum-doped mesoporous silica produced in Production Example 4 exhibited excellent antiviral activity against influenza virus, an enveloped virus. Furthermore, the slurry containing copper- and aluminum-doped mesoporous silica produced in Production Example 3 also exhibited excellent antiviral activity against feline calicivirus, a non-enveloped virus. The slurry containing aluminum-doped mesoporous silica produced in Production Example 4 also had an antiviral activity against feline calicivirus, although this was inferior to the slurry containing copper- and aluminum-doped mesoporous silica produced in Production Example 3. Neither the slurry containing copper- and aluminum-doped mesoporous silica produced in Production Example 3 nor the slurry containing aluminum-doped mesoporous silica produced in Production Example 4 was toxic to host cells or significantly reduced the susceptibility of host cells to viruses. Furthermore, as is clear from Table 2, the BSA adsorption amount was an extremely high value of 100 μg or more per mg for both the copper- and aluminum-doped mesoporous silica produced in Production Example 1 and the aluminum-doped mesoporous silica produced in Production Example 2. Furthermore, a proteolytic effect was observed in the slurry containing the copper- and aluminum-doped mesoporous silica produced in Production Example 3 (a smear band was observed in electrophoresis, indicating the presence of BSA degradation products of various molecular weights).

[0044] The antiviral activity of commercially available non-metal-doped mesoporous silica (MCM-41, manufactured by Aldrich, pore diameter: 2.1 to 2.7 nm (manufacturer's value)) was investigated in accordance with Test Example 1, and the results were as follows. As in Production Example 3, 11 g of this non-metal-doped mesoporous silica was placed in a 250 mL polypropylene pot together with 99 g of ion-exchanged water and 210 g of 2 mm diameter alumina balls, and wet-pulverized in a pot mill for 3 hours. The alumina balls were then filtered off to obtain a slurry (pH: approximately 7) containing 10 wt % non-metal-doped mesoporous silica with a median diameter of 1.17 μm. The X-ray diffraction intensity of the mesoporous silica was measured after filtering and drying the fine particles in the slurry, and the d value had a peak at a position of 3.7 nm. The slurry had an influenza virus infectivity titer of 1.0 × 10 8 PFU / mL) and feline calicivirus (infectivity titer: 2.6 × 10 8 The antiviral activity against influenza virus (PFU / mL) is shown in Table 3. As is clear from Table 3, the slurry containing non-metal-doped mesoporous silica exhibited antiviral activity against influenza virus, but the extent of this activity was slight. No antiviral activity against feline calicivirus was observed. The amount of BSA adsorbed by this non-metal-doped mesoporous silica was only 7.5 μg per mg. Furthermore, no proteolytic activity was observed in the slurry containing this non-metal-doped mesoporous silica (no smear bands were observed in electrophoresis, indicating the presence of BSA degradation products of various molecular weights). These results contrast with the antiviral activity of metal-doped mesoporous silica.

[0045] [Table 3]

[0046] Preparation Example 5: Preparation of non-porous silica doped with copper and aluminum Solution (a) was prepared by dissolving sodium silicate (sodium metasilicate nonahydrate; manufactured by Nippon Chemical Industry Co., Ltd., SiO₂ / Na₂O = 0.9–1.1 (manufacturer's value)) as a silica source in water as a solvent, while solution (b) was prepared by dissolving copper chloride, a source for doping copper into nonporous silica, and aluminum chloride, a source for doping aluminum into nonporous silica, in water as a solvent. Solutions (a) and (b) were mixed at room temperature and stirred for 5 minutes, after which hydrochloric acid was added dropwise over 30 minutes while stirring (the pH was 14 at the start of the hydrochloric acid addition and 9 at the end). After approximately 30 minutes, the resulting precipitate was filtered under suction and thoroughly washed with water until the electrical conductivity of the supernatant was below 1000 μS, after which it was dried overnight at 80°C. The collected filter cake was coarsely crushed in a mixer and then calcined at 570°C for 5 hours to obtain the desired copper- and aluminum-doped nonporous silica as a slightly bluish white powder.

[0047] The amounts of copper chloride used as a raw material for doping copper into non-porous silica, aluminum chloride used as a raw material for doping aluminum into non-porous silica, and water used as a solvent were as follows, relative to 1 mol of sodium silicate used as a silica raw material: Copper chloride: 0.0201 mol Aluminum chloride: 0.0475 mol Water for preparing solution (a): 48.6 mol Water for preparing solution (b): 90.2 mol Furthermore, 1.59 mol of hydrochloric acid was used per 1 mol of sodium silicate as the silica raw material.

[0048] The copper and aluminum doped non-porous silica obtained by the above method has a BET specific surface area of ​​18.8 m 2 / g. The copper content in the copper- and aluminum-doped nonporous silica was 1.43 wt%, and the aluminum content was 2.03 wt%. The doping of the nonporous silica with copper and aluminum was confirmed using an X-ray photoelectron spectrometer and a transmission electron microscope.

[0049] Preparation Example 6: Preparation of copper-doped non-porous silica Copper-doped silica was prepared in the same manner as in Production Example 5, except that aluminum chloride used in Production Example 5 as a raw material for doping aluminum into non-porous silica was not used, and 1.73 mol of hydrochloric acid was used per 1 mol of sodium silicate used as a silica raw material. Non Porous silica was obtained as a white powder (BET specific surface area: 4.92 m 2 / g, copper content: 1.63 wt%).

[0050] Preparation Example 7: Preparation of manganese-doped non-porous silica A manganese-doped silica was prepared in the same manner as in Production Example 5, except that manganese chloride was used as a raw material for doping manganese into non-porous silica in an amount of 0.0204 mol per 1 mol of sodium silicate as the silica raw material instead of copper chloride as the raw material for doping copper into non-porous silica, aluminum chloride was not used as a raw material for doping aluminum into non-porous silica, and 1.73 mol of hydrochloric acid was used per 1 mol of sodium silicate as the silica raw material. Non Porous silica was obtained as a white powder (BET specific surface area: 8.74 m 2 / g, manganese content: 1.13 wt%).

[0051] Preparation Example 8: Preparation of a slurry in which copper and aluminum doped non-porous silica is suspended in a dispersion medium In the same manner as in Production Example 3, 11 g of the copper- and aluminum-doped nonporous silica produced in Production Example 5 was placed in a 250 mL polypropylene pot together with 99 g of ion-exchanged water and 210 g of 2 mm diameter alumina balls, and wet-pulverized in a pot mill for 24 hours. The alumina balls were then filtered off to obtain a slurry (pH: approximately 7) containing 10 wt % copper- and aluminum-doped nonporous silica with a median diameter of 1.23 μm.

[0052] Preparation Example 9: Preparation of a slurry in which copper-doped non-porous silica is suspended in a dispersion medium A slurry (pH: approximately 7) containing 10 wt % copper-doped nonporous silica having a median diameter of 2.58 μm was obtained in the same manner as in Production Example 8, except that the copper-doped nonporous silica produced in Production Example 6 was used instead of the copper- and aluminum-doped nonporous silica produced in Production Example 5.

[0053] Preparation Example 10: Preparation of a slurry in which manganese-doped non-porous silica is suspended in a dispersion medium A slurry (pH: approximately 7) containing 10 wt % manganese-doped nonporous silica with a median diameter of 1.08 μm was obtained in the same manner as in Production Example 8, except that the manganese-doped nonporous silica produced in Production Example 7 was used instead of the copper and aluminum-doped nonporous silica produced in Production Example 5.

[0054] Test Example 2: Antiviral effect of metal-doped nonporous silica According to Test Example 1, the influenza virus infectivity titer of a slurry containing 10 wt% of the metal-doped nonporous silica prepared in Preparation Examples 8 to 10 in ion-exchanged water as a dispersion medium was 4.8 × 10 7 The antiviral activity against influenza virus (PFU / mL) was investigated (however, the contact time between the virus suspension and the specimen was 24 hours). The results are shown in Table 4. As is clear from Table 4, all of the slurries containing metal-doped nonporous silica produced in Production Examples 8 to 10 exhibited excellent antiviral activity against influenza virus, an enveloped virus. Furthermore, the slurry containing copper-doped nonporous silica produced in Production Example 8 was observed to have a proteolytic activity (a smear band was observed in electrophoresis, indicating the presence of BSA degradation products of various molecular weights).

[0055] [Table 4]

[0056] (Considerations based on the results of Test Examples 1 and 2) The mechanism by which the inorganic antiviral agent of the present invention exerts its antiviral effect is not clear at present, but it does not appear to be due to copper ions, which are known to have antiviral activity. This is because, for example, the slurry containing copper- and aluminum-doped mesoporous silica produced in Production Example 3 hardly elutes copper ions into the dispersion medium (the elution amount was 10 ppm even after leaving the silica standing for 55 days: measured using an inductively coupled plasma atomic emission spectrometer), and the antiviral effect is exerted even if the metal doped in the silica is not copper. In light of these points, the inventors believe that the inorganic antiviral agent of the present invention adsorbs viruses by protein adsorption while the metal remains chemically bonded to and incorporated into the inorganic network consisting of siloxane bonds that constitute silica, or generates reactive oxygen species in the system that attack viruses, thereby denaturing or decomposing the virus structure and irreversibly damaging its function (in fact, it has been confirmed that the inorganic antiviral agent of the present invention has the ability to decompose hemagglutinin, a surface protein of influenza viruses).

[0057] Test Example 3: Antiviral effect of articles containing metal-doped silica The copper- and aluminum-doped mesoporous silica produced in Production Example 3 was suspended in a dispersion medium to form a slurry, which was then mixed with a commercially available acrylic self-crosslinking resin binder and water. This slurry was applied to a polyester nonwoven fabric and dried at 150°C for 7 minutes to produce a nonwoven fabric coated with copper- and aluminum-doped mesoporous silica (mesoporous silica content relative to the nonwoven fabric was 20 wt%). The processed nonwoven fabric was tested for influenza virus infection (infectivity titer: 5.0 x 10) according to JIS L1922 "Testing Methods for Antiviral Activities of Textile Products." 7 PFU / mL) and feline calicivirus (infectivity titer: 1.5 × 10 7An antiviral test was conducted on the antiviral activity of the compound against enveloped and non-enveloped viruses. The results are shown in Table 5. As is clear from Table 5, the compound exhibited excellent antiviral activity against both enveloped and non-enveloped viruses.

[0058] [Table 5] [Industrial Applicability]

[0059] INDUSTRIAL APPLICABILITY The present invention has industrial applicability in that it can provide an inorganic antiviral agent that exhibits excellent antiviral activity not only against enveloped viruses but also against non-enveloped viruses.

Claims

1. An inorganic antiviral agent comprising aluminum-doped silica (as used above, "aluminum-doped silica" means silica in which aluminum is incorporated by chemical bonding into the inorganic network consisting of siloxane bonds that constitute the silica).

2. The inorganic antiviral agent according to claim 1, wherein the silica is doped with at least one metal selected from the group consisting of copper, zirconium, cobalt, manganese, and iron (in the above, "silica is doped with a metal" means that the metal is incorporated by chemical bonding into the inorganic network consisting of siloxane bonds that constitute the silica).

3. 3. The inorganic antiviral agent according to claim 2, wherein the silica is doped with copper and aluminum.

4. 4. The inorganic antiviral agent according to any one of claims 1 to 3, wherein the silica is porous silica.

5. 4. The inorganic antiviral agent according to any one of claims 1 to 3, wherein the silica is non-porous silica.

6. An article comprising the inorganic antiviral agent according to any one of claims 1 to 5.

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