Antibacterial and antiviral agents and their use

Abstract

The present invention provides an antibacterial agent or an antiviral agent based on fucoidan, the antibacterial agent or the antiviral agent being characterized by containing a silver-bonded fucoidan in which silver is bonded to a sulfate group of fucoidan, and the antibacterial agent or the antiviral agent having a good effect.

Description

Technical Field

[0001] The present invention relates to an antibacterial agent and an antiviral agent containing silver-bonded fucoidan and uses thereof. Background Art

[0002] Infectious diseases caused by bacteria or viruses have a great impact on people's lives. In order to prevent infectious diseases, the following methods are considered: people wear sanitary products such as masks, and use antibacterial or antiviral agents in places that people come into contact with.

[0003] Until now, most of the drugs used for antibacterial and antiviral treatments are alcohol-based, and sometimes a rash may occur depending on the individual.

[0004] The present inventors have been searching for safer antibacterial or antiviral agents that are derived from edible natural ingredients, and have selected fucoidan from brown algae as a candidate. It is known that fucoidan can inhibit the invasion of coronaviruses or the adhesion of coronaviruses to heparin (Non-patent Documents 1 and 2), but its effect is by no means excellent.

[0005] Prior art literature

[0006] Non-patent literature

[0007] Non-patent document 1: Sung-Kun Yim, et.al., "Inhibition of SARS-CoV-2Virus Entry by the Crude Polysaccharides of Seaweeds and Abalone Viscera In Vitro", Mar.Drugs 2021, 19, 219.

[0008] Non-patent literature 2: Paul S. Kwon, et al., "Sulfated polysaccharides effectively inhibit SARS-CoV-2 in vitro", Cell Discovery (2020) 6:50. Summary of the invention

[0009] Therefore, an object of the present invention is to provide an antibacterial agent and an antiviral agent based on fucoidan, which has a high effect.

[0010] The present inventors have conducted intensive studies to solve the above problems and have found that silver-bonded fucoidan, in which silver is bonded to the sulfate group of fucoidan, can be an excellent antibacterial agent and antiviral agent, thereby completing the present invention.

[0011] That is, the present invention relates to an antibacterial agent or an antiviral agent characterized by containing silver-bonded fucoidan in which silver is bonded to a sulfate group of fucoidan.

[0012] The present invention also relates to an antibacterial / antiviral product, characterized in that an object to be treated is treated with the above-mentioned antibacterial agent and antiviral agent.

[0013] Furthermore, the present invention relates to a silver-bonded fucoidan, characterized in that silver is bonded to a sulfate group of fucoidan.

[0014] The antibacterial agent or antiviral agent of the present invention has a high effect and is also highly safe.

[0015] Therefore, the antibacterial or antiviral agent of the present invention can be used as a countermeasure against infectious diseases by spreading it in places where people come into contact with it, or using it to treat objects such as sanitary products, or spraying it into the throat or nasal cavity, or ingesting it as food or drink. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a graph showing the results of the bacterial growth inhibitory activity measurement of Test Example 1 (in the graph, F represents fucoidan, and AgF represents silver-bonded fucoidan).

[0017] Figure 2 It is a graph showing the results of the bacterial growth inhibitory activity measurement of Test Example 2 (in the graph, F represents fucoidan, AgF represents silver-bonded fucoidan, Ag represents silver nitrate, and Ag+F represents a mixed solution of silver nitrate and fucoidan).

[0018] Figure 3 This is a diagram showing the results of the activity measurement of inhibiting the binding of coronavirus spike protein and heparin in Test Example 3 (in the diagram, AgF represents silver-bonded fucoidan).

[0019] Figure 4 It is a graph showing the results of the activity measurement for inhibiting the proliferation of Staphylococcus epidermidis in Test Example 4 (in the graph, F represents fucoidan, AgF represents silver-bonded fucoidan, Ag represents silver nitrate, and Ag+F represents a mixed solution of silver nitrate and fucoidan).

[0020] Figure 5 It is a graph showing the results of the measurement of the activity for inhibiting the proliferation of black mold in Test Example 5 (in the graph, F represents fucoidan, AgF represents silver-bonded fucoidan, Ag represents silver nitrate, and Ag+F represents a mixed solution of silver nitrate and fucoidan).

[0021] Figure 6 This is a diagram showing the results of the activity measurement for inhibiting SARS2-CoV-2 cell infection in Test Example 6 (AgF represents silver-bonded fucoidan). DETAILED DESCRIPTION

[0022] The antimicrobial agent of the present invention contains silver-bonded fucoidan in which silver is bonded to the sulfate group of fucoidan.

[0023] Fucoidan, which is the raw material of the above-mentioned silver-bonded fucoidan, is a polysaccharide unique to brown algae, and has sulfated fucose in the constituent sugar. The brown algae are not particularly limited, and examples thereof include Cladosiphonokamuranus, Undaria pinnatifida, Sargassum horneri (Turner) C. Agardh, Laminaria Japonica Areschoug, Fucus distichus, Sargassum fulvellum, etc. Among these, Okinawa Mozuku fucoidan from Okinawa Mozuku is preferred. The Okinawa Mozuku fucoidan is formed by using α1,3-bonded fucose as the main chain, bonding 1 molecule of glucuronic acid to 4 to 6 molecules of fucose, and half of the fucose is sulfated.

[0024] These fucoidans can be used without particular limitation, and those extracted by methods described in literature (M. Nagaoka, et al.: Structural study of fucoidan from Cladosiphon okamuranus TOKIDA. Gly coconjugate Journal 16: 19-26, 1999), patent (Japanese Patent No. 3920954), etc. In addition, fucoidan can be obtained by hydrolysis with an acid such as hydrochloric acid, or by purification treatment such as acid washing using an ultrafiltration membrane, electrodialysis, or an ion exchange column described in the above patents.

[0025] The molecular weight of fucoidan is not particularly limited, and is, for example, 5 to 250 kDa, preferably 20 to 200 kDa. The molecular weight is a value measured by size exclusion chromatography.

[0026] Such fucoidan can be used as a product commercially available from Yakult Pharmaceutical Industry Co., Ltd. as YAKULT FUCOIDAN or the like.

[0027] The method for bonding silver to the sulfate group of the fucoidan is not particularly limited as long as it is a method capable of bonding silver to the sulfate group of fucoidan, and examples thereof include methods using an ion exchange resin, electrodialysis, or an ultrafiltration membrane.

[0028] The fucoidan used in the above-mentioned silver bonding method is not particularly limited, and examples thereof include a solution obtained by dissolving fucoidan in water, etc. The content of fucoidan in the solution is not particularly limited, and is, for example, 0.0002 to 10% by mass (hereinafter referred to as "%"), preferably 0.1 to 8%. The pH of the solution is not particularly limited, and is 2 to 12, and preferably 4 to 10.

[0029] The silver used is not particularly limited, and examples thereof include a solution prepared by dissolving silver nitrate in water, etc. The silver content in the solution is not particularly limited, and is, for example, 0.05 μM to 20 M, preferably 0.6 μM to 15 M, in terms of silver nitrate. The pH of the solution is not particularly limited, and is 2 to 12, preferably 4 to 10.

[0030] Specifically, the method of using an ion exchange resin to bond silver to the sulfate group of fucoidan is as follows. First, a 0.6μM to 15M silver nitrate solution is added to a column filled with a cation exchange resin and allowed to react at room temperature for 0.1 to 2 hours, and then deionized water is passed through the column. On the other hand, fucoidan is dissolved in deionized water in a manner of 1 to 30 mg / mL, added to the above-mentioned column and reacted at room temperature for 0.1 to 2 hours. After the reaction, the liquid is recovered with 1 to 5 times the amount of deionized water of the reaction solution and the column. Thus, a silver-bonded fucoidan is obtained.

[0031] In order to increase the amount of silver bonded to the sulfate group of fucoidan, the concentration of the silver solution may be increased, the amount of the cation exchange resin may be increased, the reaction time may be prolonged by passing the mixture through the cation exchange resin multiple times, and stirring may be performed during the reaction.

[0032] After silver is bonded to the sulfate group of fucoidan as described above, washing such as dialysis, drying such as freeze drying and spray drying, and purification such as chromatography, ultrafiltration and two-phase partitioning are further performed.

[0033] In the silver-bonded fucoidan thus obtained, silver is bonded to the sulfate group of fucoidan. The mass ratio of fucoidan to silver is not particularly limited, but is 1:0.0001 or more, preferably 1:0.001 or more, and more preferably 1:0.01 to 0.2. It should be noted that the amount of silver bonded to the sulfate group of fucoidan can be measured, for example, by inductively coupled plasma emission spectroscopy.

[0034] Silver-bonded fucoidan has an antibacterial effect. Here, the antibacterial effect refers to the effect of inhibiting bacterial proliferation, the bactericidal effect, etc., and particularly refers to the effect of inhibiting bacterial proliferation. Inhibiting bacterial proliferation means that the bacteria do not proliferate or inhibit the proliferation rate. Specifically, when 100 μg / mL of silver-bonded fucoidan is brought into contact with bacteria, the proliferation of the number of bacteria is inhibited to less than 50%, preferably less than 90%, compared to the case where no silver-bonded fucoidan is added.

[0035] Specifically, the proliferation of inhibition bacteria refers to the proliferation of inhibition gram-negative bacteria, gram-positive bacteria and fungi. As the above-mentioned gram-negative bacteria, for example, Escherichia coli, Salmonella, Vibrio parahaemolyticus, Klebsiella pneumoniae and Pseudomonas aeruginosa etc. can be mentioned. In addition, as the above-mentioned gram-positive bacteria, for example, Staphylococcus aureus, Clostridium perfringens, Legionella, Bacillus cereus and Bacillus subtilis etc. can be mentioned. In addition, as the above-mentioned fungi, for example, Aspergillus niger, blue mold, black mold etc. can be mentioned. Among these fungi, preferably Escherichia coli, Salmonella, Staphylococcus aureus, Clostridium perfringens, Staphylococcus epidermidis, more preferably Escherichia coli, Staphylococcus epidermidis, black mold. Silver-bonded fucoidan can inhibit the proliferation of more than one of these fungi.

[0036] In addition, silver-bonded fucoidan has an antiviral effect. Here, the antiviral effect refers to the effect of inhibiting viral infection and / or the effect of inhibiting viral proliferation. Specifically, it refers to hindering the adhesion of viruses to cells to inhibit viral infection, and inhibiting viral proliferation.

[0037] Here, hindering the adhesion of viruses to cells refers to, for example, hindering the binding of molecules or receptors present on the cell surface used when the virus recognizes cells to the virus, and preferably refers to hindering the adhesion of the virus to heparan sulfate. It should be noted that heparan sulfate is present on the cell surface. Here, hindering means reducing the probability of adhesion. Specifically, in the presence of heparin, a type of heparan sulfate, and the spike protein of the virus, the binding of heparin to the spike protein can be suppressed to less than 90% when 100 μg / mL of silver-bonded fucoidan is added compared to the case where no silver-bonded fucoidan is added. As the above-mentioned virus, there is no particular limitation as long as it is a virus that adheres to heparan sulfate on the cell surface, and for example, coronaviruses, herpes viruses, etc. can be cited.

[0038] In addition, inhibiting the proliferation of viruses refers to directly acting on the viruses to hinder the proliferation of the viruses, preferably preventing the viruses from proliferating or reducing the proliferation rate. As the above-mentioned viruses, for example, coronavirus, influenza virus, norovirus, feline calicivirus, dengue virus, herpes virus, adenovirus, RS virus, rhinovirus, parainfluenza virus and the like can be cited, preferably coronavirus, influenza virus, norovirus, herpes virus and the like. Silver-bonded fucoidan can inhibit the proliferation of one or more of these viruses.

[0039] The silver-bonded fucoidan can be used as an antibacterial agent or antiviral agent because it has antibacterial and antiviral effects. The antibacterial agent and antiviral agent of the present invention can be used in the same manner as existing antibacterial agents or antiviral agents. In addition, since the silver-bonded fucoidan has the effect of adhering to viruses and inactivating them, the antiviral effect is very good.

[0040] Furthermore, the antibacterial agent and antiviral agent of the present invention may contain, for example, fragrance, ethanol, etc., as long as the effects of the present invention are not inhibited.

[0041] The dosage form of the antibacterial agent and antiviral agent of the present invention is not particularly limited, and examples thereof include liquid preparations, powders, granules, tablets, granules, capsules, etc. Among these, liquid preparations are preferred.

[0042] More specifically, the antibacterial agent and antiviral agent of the present invention can be spread, for example, in places where people come into contact with the body. The places where people come into contact with the body are not particularly limited, and examples thereof include door handles, tables, chairs, partitions, toys, training equipment, buttons of elevators, etc. The amount of the antibacterial agent or antiviral agent used in this case is not particularly limited, and for example, 1 cm 2 The amount of the compound (calculated as silver-bonded fucoidan) is 1 ng to 200 μg, preferably 2 ng to 100 μg. A sprayer or the like can be used for the dispersion.

[0043] In addition, the antibacterial agent and antiviral agent of the present invention are used to treat, for example, sanitary products and other processed objects. The method of using the antibacterial agent and antiviral agent of the present invention to treat sanitary products and other processed objects is not particularly limited, and for example, coating, covering and the like methods can be cited. Examples of sanitary products include masks, wet wipes, etc. The amount of antibacterial agent or antiviral agent treated in this case is not particularly limited, and for example, per 1 cm 2 The concentration is 1 ng to 200 μg, preferably 2 ng to 100 μg. The treated object has antibacterial and antiviral effects. The treatment can be performed using a sprayer, a brush, or the like.

[0044] In addition, the antibacterial agent and antiviral agent of the present invention can be sprayed into the throat or nasal cavity, or used in contact with the throat like a mouthwash. The amount of the antibacterial agent or antiviral agent used in this case is not particularly limited, for example, 0.1 μg to 20 mg per 1 ml, preferably 0.5 μg to 15 mg, more preferably 10 μg to 10 mg, and particularly preferably 100 μg to 5 mg. Spraying can be performed using a sprayer, etc.

[0045] In addition, the antibacterial agent and antiviral agent of the present invention can also be used in food and beverages such as lozenges, maltose, beverages, jellies, etc. The amount of the antibacterial agent or antiviral agent used in this case is not particularly limited, for example, 0.1 μg to 5 mg per 1 ml or 1 g, preferably 0.5 μg to 5 mg, more preferably 10 μg to 5 mg, and particularly preferably 100 μg to 5 mg.

[0046] When the antibacterial agent and antiviral agent of the present invention are used in tea beverages, silver-bonded fucoidan may be used instead of fucoidan in tea beverages containing fucoidan as described in, for example, Japanese Patent No. 3579597.

[0047] As described above, the antibacterial agent and antiviral agent of the present invention can be used as a countermeasure against infectious diseases.

[0048] Example

[0049] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0050] Example 1

[0051] Preparation of silver-bonded fucoidan:

[0052] 2M silver nitrate solution was added to a column filled with a cation exchange resin (Dowex50, FUJIFI LM Wako Chemical) and reacted at room temperature for 1 hour, and then deionized water was passed through the column. Okinawa Mozuku Fucoidan (YAKULT FUCOIDAN, Yakult Pharmaceutical Industry, molecular weight 79kDa) was dissolved in deionized water in a manner of 10mg / mL, added to the above-mentioned column and reacted at room temperature for 1 hour. After the reaction, the deionized water of 5 times the amount of the reaction solution and the column was recovered and lyophilized to obtain silver-bonded silver-bonded fucoidan in which silver was bonded to a sulfate group. It should be noted that the content of silver in the silver-bonded fucoidan was determined by inductively coupled plasma emission spectroscopy, and the result was 0.037mg per 1mg.

[0053] Test Example 1

[0054] Determination of the activity of inhibiting the proliferation of Escherichia coli:

[0055] The silver-bonded fucoidan (AgF) prepared in Example 1 was dissolved in tryptic soy broth (TSB) at a final concentration of 10 μg / mL, 100 μg / mL, and 1000 μg / mL, and 5% of the culture solution obtained by culturing Escherichia coli (E. coli K12 strain) in TSB for 24 hours was added thereto. It was cultured at 37°C, and the OD600nm at each time was measured to analyze the activity of inhibiting the proliferation of E. coli. In addition, as a comparison, the activity of inhibiting the proliferation of E. coli was also analyzed for the addition of 10 μg / mL, 100 μg / mL, 1000 μg / mL, and 10000 μg / mL of Okinawa Mozuku Fucoidan (F) (YAKULT FUCOIDAN, manufactured by Yakult Pharmaceutical Industry, molecular weight 79 kDa). These results are shown in Figure 1 .

[0056] With respect to Escherichia coli, Okinawa Mozuku fucoidan began to show growth inhibitory activity at 10000 μg / mL, whereas silver-bonded fucoidan showed growth inhibitory activity at 10 μg / mL.

[0057] Test Example 2

[0058] Determination of the activity of inhibiting the proliferation of Escherichia coli:

[0059] Silver-bonded fucoidan (AgF) prepared in Example 1, Okinawa mozuku fucoidan (F) (YAKULTFUCOIDAN, manufactured by Yakult Pharmaceutical Industry, molecular weight 79 kDa), silver nitrate (Ag), and a mixed solution of Okinawa mozuku fucoidan and silver nitrate (Ag+F) were used at the concentrations shown in Table 1, and the activity of inhibiting the growth of Escherichia coli was analyzed in the same manner as in Test Example 1. These results are shown in Figure 2 .

[0060] [Table 1]

[0061]

[0062] For Escherichia coli, silver-bonded fucoidan showed a significantly higher growth inhibitory effect than the same amount of silver and fucoidan alone or their mixture.

[0063] Test Example 3

[0064] Determination of the activity of inhibiting coronavirus adhesion:

[0065] The silver-bonded fucoidan (AgF) prepared in Example 1 was used to confirm the effect of inhibiting the binding of coronavirus to heparan sulfate. Biacore (Cytiva) was used to analyze the effect of inhibiting the binding of SARS-CoV-2 spike protein and heparin (HP, a type of heparan sulfate). For HP immobilized on the small piece, 100 or 500 ng / mL of AgF was added to the SARS-CoV-2 spike protein (100nM), and the binding amount (Resonance Unit) was measured. As a positive control, a system using HP instead of AgF was performed. Each system was performed 3 times, and the average results are shown in Figure 3 (The bars in the graph represent standard deviation).

[0066] according to Figure 3 , it was confirmed that AgF hinders the binding of coronavirus spike protein and HP.

[0067] Example 2

[0068] Production of antibacterial and antiviral agents:

[0069] The antibacterial and antiviral agent was prepared by mixing the components of the following formulation.

[0070] (Element)

[0071] Silver-bonded fucoidan (the substance prepared in Example 1) 0.01%

[0072] Water 99.99%

[0073] The antibacterial and antiviral agent was sprayed onto the door handle using a sprayer and allowed to dry.

[0074] Example 3

[0075] Production of antibacterial and antiviral agents:

[0076] The antibacterial and antiviral agent was prepared by mixing the components of the following formulation.

[0077] (Element)

[0078] Silver-bonded fucoidan (the substance prepared in Example 1) 0.01%

[0079] Water 99.98%

[0080] Fragrance 0.01%

[0081] The antibacterial and antiviral agent is uniformly sprayed onto the mask using a sprayer and then dried to produce an antibacterial and antiviral mask.

[0082] Example 4

[0083] Production of antibacterial and antiviral agents:

[0084] The antibacterial and antiviral agent was prepared by mixing the components of the following formulation.

[0085] (Element)

[0086]

[0087]

[0088] The antibacterial and antiviral agent is sprayed into the throat or nasal cavity using a sprayer.

[0089] Example 5

[0090] Production of beverages containing antibacterial and antiviral agents:

[0091] The components of the following formulation were mixed to produce a beverage containing an antibacterial / antiviral agent.

[0092] (Element)

[0093] Silver-bonded fucoidan (the substance prepared in Example 1) 0.01%

[0094] Tea extract 99.99%

[0095] Example 6

[0096] Preparation of silver-bonded fucoidan:

[0097] (1) Cleaning of cation exchange resin

[0098] After passing 1M hydrochloric acid 5 times the volume of the resin, ultrapure water 4 times the volume, and 1M sodium hydroxide solution 3 times the volume in the order of 60 mL of a cation exchange resin (strong acid cation exchange resin No. 4, crosslinking degree 8%, 50-100 mesh, H-shaped, FUJIFILM Wako 355-4568), 3 times the volume of 1M sodium hydroxide solution was added, and the mixture was reacted at 65° C. for 2 hours while being constantly stirred with a glass rod. This operation was repeated once.

[0099] (2) Resin activation and silver ionization

[0100] After washing, the resin is suspended in 4 times the amount of ultrapure water. Through the washed resin, 3 times the amount of 1M sodium hydroxide solution, 3 times the amount of ultrapure water, 1 times the amount of 4M hydrochloric acid, and 3 times the amount of 1M hydrochloric acid are passed in order to activate the resin, and 5 times the amount of ultrapure water is passed (until the pH becomes the same as that of ultrapure water) for washing. Add 2.5 times the amount of 2M silver nitrate solution to the activated resin, shake at 25°C until the resin moves and pass the liquid for 60 hours. Next, after passing 3 times the amount of 1M silver nitrate solution through the column, pass 5 times the amount of ultrapure water for washing, thereby preparing a silver ionized resin.

[0101] (3) Adding silver ions to fucoidan

[0102] Okinawa Mozuku Fucoidan (YAKULT FUCOIDAN, Yakult Pharmaceutical Industry, molecular weight 79kDa) was dissolved in ultrapure water to 10mg / mL, filtered with a 3μm filter (ADVANTEC, C300A047A), and dialyzed for 3 days with a dialysis membrane (MWCO7000, Spectrum Laboratories Spectra / Por No.1 132655) while replacing ultrapure water, and the fucoidan solution was used for the test. The silver-ionized resin and fucoidan solution were moved to a culture medium bottle, and reacted for 60 hours at 25°C while shaking to the extent that the resin moved. The resin and fucoidan solution were moved to a column, and after passing 3 times the amount of ultrapure water, the liquid was freeze-dried to obtain a freeze-dried product of silver-bonded fucoidan. It should be noted that the silver content in the silver-bonded fucoidan was measured by inductively coupled plasma emission spectroscopy, and the result was 0.137mg per 1mg.

[0103] Test Example 4

[0104] Determination of the activity of inhibiting the proliferation of Staphylococcus epidermidis:

[0105] Staphylococcus epidermidis ATCC 14990: standard strain) was inoculated into 3 mL of TSB and pre-cultured at 37°C. Silver-bonded fucoidan (AgF) prepared in Example 6, Okinawa Mozuku fucoidan (F) (YAKULT FUCOIDAN, manufactured by Yakult Pharmaceutical Industry, molecular weight 79 kDa) and silver nitrate (Ag) were prepared in pure water to concentrations of 1 mg / mL, 10 mg / mL and 2 mg / mL, respectively, and stirred at 4°C for 24 hours to dissolve. After filtering them with a 0.45 μm filter, 50, 100 and 200 μg / mL of silver-bonded fucoidan prepared by dilution with TSB, fucoidan solutions equivalent to the silver-bonded fucoidan at each concentration, silver (silver nitrate) solutions, and mixed solutions of silver nitrate and fucoidan (Table 2) were mixed with 10 μL of the pre-culture solution. Thereafter, the plate was sealed with high oxygen permeability (BEM-1, DIVERSIFIED BIOTECH), cultured at 37°C, and turbidity (600 nm) was measured over time (n=3, Infinite M200 PRO, Tecan). The condition in which only the pre-culture solution was mixed with TSB was used as a control.

[0106] [Table 2]

[0107]

[0108] The inhibitory effect of silver-bonded fucoidan on the proliferation of Staphylococcus epidermidis at any concentration was higher than that of fucoidan solution equivalent to silver fucoidan, silver (silver nitrate) solution, and the mixed solution of silver nitrate and fucoidan ( Figure 4 It is believed that silver-bonded fucoidan exhibits a proliferation inhibitory effect on Staphylococcus epidermidis at a concentration of 50 μg / mL or more, and that silver ions are ionically bonded to fucoidan, thereby exhibiting a synergistic effect on Staphylococcus epidermidis at least at a concentration of 50 to 200 μg / mL.

[0109] Test Example 5

[0110] Determination of the activity of inhibiting the proliferation of black mold:

[0111] The black mold (Cladosporium cladosporioidesNBRC 6368) obtained from the (unique) product evaluation technology base institution was inoculated into potato dextrose broth medium (potato dextrose agar; PDA, Nippon Pharmaceutical Co., Ltd.) and cultured at 25°C for 22 days. After one subculture, it was further cultured for 26 days. The growing black mold was taken out together with the PDA and immersed in a protective medium (10% glycerol, 5% trehalose solution) to prepare a frozen stock solution. Next, the frozen stock solution was thawed, inoculated into Sabouraud agar medium (Remel) and cultured at 25°C for 7 days. The surface of the growing black mold was scraped with a spreading stick (bacteria spreader) and suspended in Sabouraud liquid medium (Oxoid), filtered with a funnel equipped with a Kimwipe, and the obtained filtrate was used as a spore liquid. The number of spores in the spore liquid was counted using a hemacytometer to become 10 7 The cells were prepared in Sabouraud liquid medium at a rate of 1 cell / mL.

[0112] After dissolving in pure water in the same manner as in Test Example 4, 190 μL of each of 10, 20, and 40 μg / mL silver-bonded fucoidan prepared by diluting with Sabouraud's liquid medium instead of TBS, a fucoidan solution corresponding to each concentration of silver-bonded fucoidan, a silver (silver nitrate) solution, and a mixed solution of silver and fucoidan (Table 3) were added to the spore solution (spore count 10 7 After mixing with 10 μL of spore solution (10 μL / mL), the plate was sealed with high oxygen permeability (BEM-1, DIVERSIFIED BIOTECH), cultured at 25°C, and the turbidity (600 nm) was measured over time (n=3, Infinite M200 PRO, Tecan), and the turbidity change from the start of culture was calculated. The condition of mixing spore solution with Sabouraud liquid medium was used as a control.

[0113] [Table 3]

[0114]

[0115] For black mold, no proliferation inhibition was observed for 10 μg / mL silver-bonded fucoidan, silver nitrate solution equivalent to silver-bonded fucoidan, and a mixture of silver nitrate and fucoidan, but 20 μg / mL silver-bonded fucoidan showed a higher proliferation inhibition effect than silver nitrate solution equivalent to silver-bonded fucoidan and a mixture of silver nitrate and fucoidan ( Figure 5 ). In addition, 40 μg / mL of silver-bonded fucoidan, a silver nitrate solution equivalent to silver-bonded fucoidan, and a mixture of silver nitrate and fucoidan all inhibited proliferation after 60 hours of culture. The fucoidan solution did not inhibit proliferation at any concentration. It is believed that silver-bonded fucoidan exhibits a proliferation inhibitory effect on black mold at 20 μg / mL or above, and that silver ions are ionically bonded to fucoidan, thereby exhibiting a synergistic effect on black mold at least at 20 to 40 μg / mL.

[0116] Test Example 6

[0117] Determination of the activity of inhibiting SARS2-CoV-2 infection of cells:

[0118] (1) Preparation of test substances

[0119] The silver-bonded fucoidan prepared in Example 6 was prepared at 100 μg / mL with Milli Q water, filtered through a 0.45 μm filter, and then subjected to experiments by 10-fold serial dilution (100 ng / ml, 1 μg / ml, 10 μg / ml).

[0120] (2) Evaluation of viral cell infection using luciferase luminescence

[0121] According to a previous report (Tani et al. Virol J (2021) 18: 16), a pseudotyped virus (SARS2-pv) was prepared in which the SARS-CoV-2 spike protein was encapsulated by vesicular stomatitis virus (VSV) with the gene encoding the envelope protein deleted by the insertion of the luciferase gene. Cell lines stably expressing angiotensin-converting enzyme 2 (ACE2) were cultured at 2×10 4The cells / well were inoculated in a 96-well white / clear bottom plate (96well White / ClearBottom Plate; Thermo Fisher Scientific). The next day, 10 μL of the test substance was mixed with 10 μL of SARS2-pv, reacted at room temperature for 5 minutes, and then added to the culture supernatant of the cells. After 24 hours of culture, 20 μL / well of PicaGene BrillianStar-LT luminescent reagent (Toyo B-Net) was added, and after standing for 5 minutes, the luminescence generated by luciferase was measured using Spectramax M5e (Molecular Devices). The effect of hindering cell infection by SARS2-pv is expressed by the ratio when the luminescence intensity of the wells mixed with SARS2-pv and solvent (Milli Q water) is set to 100%.

[0122] (3) Cell viability assay (MTS)

[0123] In a 96-well plate, 1 to 2 × 10 4 Cells were seeded in cells / well, and 10 μL of the test substance was added the next day. After culturing for 24 hours, 10 μL / well of CellTiter Aqueous One Solution Cell Proliferation Assay (Promega) was added, and the absorbance at 490 nm was measured after reacting in a CO2 incubator at 37°C for 1 hour. The wells in which only the test substance was added to the culture medium were used as the background, and the background was subtracted for each test substance. The value of the MTS detection is expressed by the ratio when the value of the well with the solvent added is set to 100%.

[0124] It was found that silver-bonded fucoidan showed an inhibitory effect on SARS-CoV-2 cell infection in a concentration-dependent manner at 100 ng / ml to 10 μg / ml ( Figure 6 ). In addition, no cytotoxicity was observed within this range.

[0125] Industrial Applicability

[0126] The antibacterial agent and antiviral agent of the present invention can be used as a countermeasure against infectious diseases caused by bacteria and viruses.

Claims

1. An antibacterial agent, characterized in that Contains silver-bonded fucoidan, in which silver is bonded to the sulfate group of fucoidan.

2. The antibacterial agent according to claim 1, wherein Fucoidan is from Okinawa Mozuku.

3. The antibacterial agent according to claim 1 or 2, which inhibits the proliferation of Gram-negative bacteria, Gram-positive bacteria or fungi.

4. The antibacterial agent according to any one of claims 1 to 3, wherein The mass ratio of fucoidan to silver is 1:0.0001 or more.

5. An antiviral agent, characterized in that Contains silver-bonded fucoidan, in which silver is bonded to the sulfate group of fucoidan.

6. The antiviral agent according to claim 5, wherein Fucoidan is from Okinawa Mozuku.

7. The antiviral agent according to claim 5 or 6, which inhibits the adhesion of viruses to heparan sulfate. The antiviral agent according to claim 5 or 6, which inhibits viral proliferation.

9. The antiviral agent according to any one of claims 5 to 8, wherein The mass ratio of fucoidan to silver is 1:0.0001 or more.

10. An antibacterial and antiviral product, characterized in that: The object to be treated is treated with the antibacterial agent according to any one of claims 1 to 4 and the antiviral agent according to any one of claims 5 to 9.

11. A silver-bonded fucoidan, characterized in that: It is made of silver bonded to the sulfate group of fucoidan.

12. The silver-bonded fucoidan according to claim 11, wherein Fucoidan is from Okinawa Mozuku.

13. The silver-bonded fucoidan according to claim 11 or 12, wherein The mass ratio of fucoidan to silver is 1:0.0001 or more.

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