Virus aggregation agent, virus inhibition agent and virus removal method

Abstract

To provide a virus aggregation agent and a virus removal method that enable the removal of viruses through general filters, and a virus inhibition agent that exhibits an increased virus inhibition rate compared to the sole use of PQQ.SOLUTION: The present invention provides a virus aggregation agent containing pyrroloquinoline quinone or the like, a virus inhibition agent containing pyrroloquinoline quinone or the like and ascorbic acid or the like, and a virus removal method including a first step for adding pyrroloquinoline quinone or the like to a virus-containing solution.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a virus agglutinating agent, a virus inhibitor, and a method for removing viruses. [Background technology]

[0002] Viruses such as coronaviruses are known to infect not only humans, but also animals such as cats, dogs, and horses. For example, seven types of coronaviruses that infect humans have been identified to date, and coronaviruses that infect cats and other animals have also been identified.

[0003] These viruses are sometimes called filterable pathogens because their size is around 20 to 100 nm and they cannot be sterilized by filtration using standard filters, which is possible with bacteria.

[0004] Therefore, there is a need to develop methods for removing these viruses and antiviral agents.

[0005] As a method for removing viruses, a method of removing viruses using a filter having pores smaller than virus particles has been reported (see Patent Documents 1 and 2). In addition, a virus removal filter including a substrate having a virus capture ability or virus adsorption ability has also been proposed (see Patent Documents 3 and 4).

[0006] On the other hand, as an antiviral agent, pyrroloquinoline quinone (hereinafter sometimes referred to as PQQ) has been reported to have inhibitory activity against viral reverse transcriptase (see Patent Document 5). It has also been reported that PQQ may suppress inflammation caused by coronavirus (see Non-Patent Document 1). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Publication No. 7-229827 [Patent Document 2] International Publication No. 2016 / 031834 [Patent Document 3] Japanese Patent Application Publication No. 11-5006 [Patent Document 4] Special Publication No. 2003-505227 [Patent Document 5] Japanese Patent Application Publication No. 1-283221 [Non-patent literature]

[0008] [Non-Patent Document 1] Alberto Boretti, PQQ Supplementation and SARS-CoV-2 Spike Protein-Induced Heart Inflammation, Nat Prod Commun. 2022 Mar 8;17(3):1934578X221080929. doi: 10.1177 / 1934578X221080929. Summary of the Invention [Problem to be solved by the invention]

[0009] However, filters having pores smaller than virus particles are special and not easily available. In addition, filters containing a base material having virus capture or removal capabilities are also special and not easily available. These filters have the disadvantage of being very expensive because they are special and uncommon.

[0010] Moreover, the PQQ antiviral agent described in Patent Document 5 does not have sufficient inhibitory activity.

[0011] Furthermore, there is no known method for removing viruses using PQQ.

[0012] Therefore, there is a need for a method that can remove viruses using relatively inexpensive and readily available general filters with pore sizes of 0.1 to 2 micrometers, and that can be achieved using non-toxic and inexpensive materials.

[0013] Furthermore, there is a demand for compositions that can enhance the virus-inhibiting activity of PQQ, which is insufficient in that activity.

[0014] The object of the present invention is to provide a virus flocculating agent and a virus removal method that enable removal of viruses using a general filter, as well as a virus inhibitor having an improved virus inhibition rate compared to the use of PQQ alone. [Means for solving the problem]

[0015] In order to achieve the above object, the present invention provides the following virus agglutinating agent, virus inhibitor, and virus removal method.

[0016] [1] A virus aggregating agent comprising pyrroloquinoline quinone, its derivatives and / or salts thereof. [2] The virus aggregating agent according to [1], further comprising ascorbic acid and / or a salt thereof. [3] The virus aggregating agent described in [2] above, further comprising one or more amino acids selected from acidic amino acids, neutral amino acids, and salts thereof. [4] The virus aggregating agent according to [3], which contains the acidic amino acid and contains glutamic acid as the acidic amino acid. [5] The virus aggregating agent according to [3], comprising the neutral amino acid, and including glycine as the neutral amino acid. [6] The virus coagulant according to [2], wherein the mass ratio of the total content of pyrroloquinoline quinone, its derivatives and / or salts thereof to the total content of ascorbic acid and / or its salts is 1:[1 to 100]. [7] The virus coagulant according to [3], wherein the mass ratio of the total content of pyrroloquinoline quinone, its derivatives and / or salts thereof:the total content of ascorbic acid and / or salts thereof:the total content of the one or more amino acids is 1:[1 to 100]:[1 to 100]. [8] The virus aggregating agent according to any one of [1] to [7] above, wherein the virus is a coronavirus. [9] The virus coagulant according to any one of the above [1] to [7], which is used as a pretreatment agent for removing viruses.

[10] A virus inhibitor comprising pyrroloquinoline quinone, a derivative thereof and / or a salt thereof, and ascorbic acid and / or a salt thereof.

[11] The virus inhibitor according to

[10] , further comprising one or more amino acids selected from acidic amino acids, neutral amino acids, and salts thereof.

[12] The virus inhibitor according to

[11] , which contains the acidic amino acid and includes glutamic acid as the acidic amino acid.

[13] The virus inhibitor according to

[11] , which contains a neutral amino acid and includes glycine as the neutral amino acid.

[14] The virus inhibitor according to

[10] , wherein a mass ratio of the total content of pyrroloquinoline quinone, its derivative, and / or a salt thereof:the total content of ascorbic acid and / or a salt thereof is 1:[1 to 10]0.

[15] The virus inhibitor according to

[11] , wherein a mass ratio of the total content of pyrroloquinoline quinone, derivatives thereof, and / or salts thereof:the total content of ascorbic acid and / or salts thereof:the total content of the one or more amino acids is 1:[1 to 100]:[1 to 100].

[16] The virus inhibitor according to any one of

[10] to

[15] above, wherein the virus is a coronavirus.

[17] A method for removing viruses, comprising a first step of adding pyrroloquinoline quinone, a derivative thereof and / or a salt thereof to a solution containing the viruses.

[18] The virus removal method described in

[17] , further comprising a filtration step of filtering the solution through a filter having a pore size of 0.1 to 2 micrometers after the first step.

[19] The virus removal method described in

[18] above, comprising a second step of adding ascorbic acid and / or a salt thereof to the solution prior to the filtration step.

[20] The method for removing viruses described in

[19] above, comprising a third step of adding one or more amino acids selected from acidic amino acids, neutral amino acids, and salts thereof to the solution before the filtration step. Effect of the Invention

[0017] According to the present invention, it is possible to provide a virus agglutinant and a virus removal method that enable viruses to be removed using a general filter, as well as a virus inhibitor that has an improved virus inhibition rate compared to the use of PQQ alone. [Brief description of the drawings]

[0018] [Figure 1] FIG. 11 is a particle size distribution diagram of Comparative Example 2. [Diagram 2] FIG. 13 is a particle size distribution diagram of Example 8. [Diagram 3] FIG. 13 is a particle size distribution diagram of Example 9. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Hereinafter, the embodiments of the present invention will be described in detail, however, the present invention is not limited to these, and various modifications are possible without departing from the gist of the present invention.

[0020] [Virus aggregating agent] A virus aggregating agent according to an embodiment of the present invention (hereinafter referred to as "the present embodiment") contains pyrroloquinoline quinone (PQQ), a derivative thereof, and / or a salt thereof.

[0021] The virus agglutinating agent according to the present embodiment has a virus agglutinating effect of agglutinating virus particles. Here, the virus agglutinating effect may be an effect of agglutinating part or all of the virus particles in a virus-containing liquid. The virus agglutinating effect can remove part or all of the viruses from the virus-containing liquid by filtration using a filter having a general pore size (0.1 to 2 μm), and the viruses in the filtrate can be reduced. According to a preferred embodiment, for example, in a virus agglutination test performed in the examples described below, the viruses can be reduced by 3% or more by filtration, according to a more preferred embodiment, the viruses can be reduced by 5% or more by filtration, and according to a further preferred embodiment, the viruses can be reduced by 7% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, or 55% or more by filtration.

[0022] The virus to be aggregated is not particularly limited. Among viruses, coronaviruses are preferred as the target of aggregation. Coronaviruses that infect humans (including the causative virus of COVID-19) as well as coronaviruses that infect animals, such as feline coronavirus, are suitable as the target of aggregation. Four types of viruses that cause relatively mild respiratory diseases such as colds are known: HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1. Three types of viruses that cause severe respiratory diseases are known: SARS-CoV (the causative virus of SARS), MERS-CoV (the causative virus of MERS), and SARS-CoV-2 (the causative virus of COVID-19), and these viruses are also targets of aggregation.

[0023] The PQQ used in this embodiment is a compound having the chemical structure shown in formula (I) below. [ka]

[0024] PQQ may be used in the form of a derivative. The derivative of PQQ can be obtained by, for example, esterifying, acylating, or silylating PQQ. In particular, a PQQ ester obtained by esterifying PQQ is preferred, and examples of preferred examples include trimethyl ester PQQ (formula (II) below) obtained by trimethyl esterifying PQQ and dimethyl ester PQQ obtained by dimethyl esterifying PQQ. Furthermore, triethyl ester PQQ, tripropyl PQQ, and tributyl PQQ are also preferred. [ka]

[0025] A preferred example of a PQQ derivative is imidazopyrroloquinoline (IPQ) (formula (III) below) obtained by reacting PQQ with an amino acid such as glycine. [ka]

[0026] PQQ and its derivatives may be used in the form of a salt. The salts of PQQ and its derivatives are formed by forming salts with alkali metal ions or the like in one to three of the carboxyl groups in the above formula. The salts of PQQ and its derivatives are not particularly limited, but for example, ammonium salts, alkali metal salts such as sodium and potassium, and alkaline earth metal salts such as calcium and magnesium are preferred. Sodium salts are particularly suitable, and monosodium salts, disodium salts, and trisodium salts can be used. In particular, pyrroloquinoline quinone disodium salt, which is a disodium salt, is easy to obtain and registered as a food, making it easy to use. The disodium salt of PQQ is represented by the following formula (IV): Preferably, it is represented by the following formula (V): [ka] [ka]

[0027] PQQ, its derivatives and their salts may be used in the form of a solvate. Examples of the solvate include hydrates, preferably monohydrates to trihydrates, more preferably trihydrates. In addition, the solvate may include a crystallization solvent.

[0028] PQQ is usually provided in the oxidized form shown in formula (I) above, but the oxidized PQQ can be easily reduced to generate the reduced PQQ. Therefore, PQQ may be in the reduced form, and the reduced PQQ is more preferable than the oxidized PQQ as a virus agglutinant. Therefore, the reduced PQQ, its derivatives and their salts can also be used. The reduced PQQ is shown in formula (VI) below. [ka]

[0029] Hereinafter, when simply referring to "PQQ", unless otherwise specified, this includes PQQ (oxidized and reduced forms), its derivatives, their salts, and their solvates.

[0030] PQQ can be produced by organic synthesis or fermentation, and is not particularly limited in the present invention. Both the organic synthesis and fermentation production methods can be carried out by known production methods. Commercially available products can also be used.

[0031] PQQ has been reported to have many physiological functions in the functional food field and its safety has been confirmed, so it can be said that the virus aggregating agent of this embodiment has no cytotoxicity and is sufficiently safe for animals such as humans.

[0032] The virus agglutinating agent according to the present embodiment preferably further contains ascorbic acid and / or a salt thereof. By further containing ascorbic acid and / or a salt thereof in addition to PQQ, the virus agglutinating effect can be enhanced. That is, the rate at which viruses can be removed from a virus-containing liquid by filtration using a filter having a general pore size (0.1 to 2 μm) can be increased, and the rate at which viruses are reduced in the filtrate can be increased.

[0033] Suitable ascorbate salts include, for example, sodium ascorbate and calcium ascorbate, and more preferably sodium ascorbate, preferably of the grade used for food products.

[0034] Ascorbic acid and / or a salt thereof may be mixed with PQQ before being added to a virus-containing liquid to form a virus aggregant, or PQQ and ascorbic acid and / or a salt thereof may be added to a virus-containing liquid, respectively, and then mixed in the liquid to form a virus aggregant. Thus, the virus aggregant according to this embodiment includes not only an embodiment in the form of a composition in which PQQ and ascorbic acid and / or a salt thereof are mixed, but also an embodiment in the form of a kit product in which each of the components is manufactured and sold as a set.

[0035] The virus agglutinating agent according to the present embodiment preferably further contains one or more amino acids selected from acidic amino acids, neutral amino acids, and salts thereof. By further containing the amino acid in addition to PQQ, the virus agglutinating effect can be enhanced. That is, the rate at which viruses can be removed from a virus-containing liquid by filtration using a filter having a general pore size (0.1 to 2 μm) can be increased, and the rate at which viruses are reduced in the filtrate can be increased. The above amino acids are more preferably used in combination with ascorbic acid and / or a salt thereof.

[0036] As the amino acid, isoleucine, leucine, methionine, phenylalanine, threonine (threonine), valine, tyrosine, cysteine, aspartic acid, asparagine, serine, glutamic acid, glutamine, proline, glycine, and alanine can be suitably used. Preferably, it is an acidic amino acid. Specifically, it is aspartic acid and glutamic acid. More preferably, it is glutamic acid. It is also preferable to contain a neutral amino acid, and it is particularly preferable to contain glycine. It may also contain a basic amino acid such as lysine, tryptophan, arginine, or histidine, but it is preferable not to contain it.

[0037] The salt of the amino acid is not particularly limited, and examples thereof include salts with alkali metals such as sodium and potassium, salts with alkaline earth metals such as calcium and magnesium, and salts with organic amines such as ammonium salts, aluminum salts, zinc salts, triethylamine, ethanolamine, morpholine, pyrrolidine, piperidine, piperazine, dicyclohexylamine, etc., for acidic groups such as carboxyl groups. Examples of salts with inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, and hydrobromic acid, salts with organic carboxylic acids such as acetic acid, citric acid, benzoic acid, maleic acid, fumaric acid, tartaric acid, succinic acid, tannic acid, butyric acid, hybenzic acid, pamoic acid, enanthic acid, decanoic acid, teoclic acid, salicylic acid, lactic acid, oxalic acid, mandelic acid, and malic acid, and salts with organic sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid, for basic groups such as amino groups.

[0038] The above amino acid may be mixed with PQQ and ascorbic acid and / or a salt thereof before being added to a virus-containing liquid to form a virus flocculant, or PQQ, ascorbic acid and / or a salt thereof, and the above amino acid may be added to a virus-containing liquid, and then mixed in the liquid to form a virus flocculant. Thus, the virus flocculant according to this embodiment includes not only an embodiment in the form of a composition in which PQQ, ascorbic acid and / or a salt thereof, and the above amino acid are mixed, but also an embodiment in the form of a kit product in which each of them is manufactured and sold as a set.

[0039] The virus aggregating agent according to the present embodiment may contain PQQ in an amount effective for aggregating the virus, and the amount is not limited. For example, the amount may be such that the PQQ concentration after addition to the virus-containing liquid is 0.1 μM to 500 μM. The amount is preferably such that the concentration is 5 to 100 μM.

[0040] The content of ascorbic acid and / or a salt thereof in the virus agglutinating agent is preferably such that the mass ratio of the total content of pyrroloquinoline quinone, its derivatives and / or salts thereof to the total content of ascorbic acid and / or a salt thereof is 1:[1 to 100]. The mass ratio is more preferably 1:[1 to 20], and further preferably 1:[2 to 10].

[0041] The content of the amino acid in the virus flocculant is preferably such that the mass ratio of the total content of pyrroloquinoline quinone, its derivatives and / or salts thereof: the total content of ascorbic acid and / or its salts: the total content of the one or more amino acids is 1:[1-100]:[1-100]. The mass ratio is more preferably 1:[1-20]:[1-20], and further preferably 1:[2-10]:[2-10].

[0042] The virus agglutinating agent according to the present embodiment preferably further contains one or more selected from calcium chloride, calcium acetate, calcium carbonate, and calcium hydroxide. By further containing calcium chloride or the like in addition to PQQ, the virus agglutinating effect can be enhanced. That is, the rate at which viruses can be removed from a virus-containing liquid by filtration using a filter having a general pore size (0.1 to 2 μm) can be increased, and the rate at which viruses are reduced in the filtrate can be increased.

[0043] The virus agglomerating agent of this embodiment can be used as a virus removal pretreatment agent that performs pretreatment (specifically, agglomeration of virus particles) in a step preceding the virus removal step, for example in the first step of the virus removal method described below, to enable virus removal in the next or subsequent step.

[0044] [Virus removal method] The virus removal method according to the present embodiment includes a first step of adding pyrroloquinoline quinone, its derivatives, and / or salts thereof to a solution containing viruses. After the addition, these are mixed to aggregate some or all of the virus particles.

[0045] The virus removal method according to the present embodiment preferably further comprises, after the first step, a filtration step of filtering the virus-containing solution to which PQQ has been added through a filter having a pore size of 0.1 to 2 μm. The pore size of the filter is preferably 0.1 to 1.5 μm, more preferably 0.1 to 1 μm, and even more preferably 0.1 to 0.5 μm. As a general filter, a pore size of 0.2 μm to 0.5 μm is inexpensive and easy to use. The material of the filter can be selected according to the purpose. It may be made of either ceramic or polymer. Preferably, PTFE (polytetrafluoroethylene), PVDF (polyvinylidene fluoride), PES (polyethersulfone), nylon, cellulose mixed ester, polypropylene, and cellulose are easy to use and are preferred.

[0046] Filtration can be done with either a pump or a syringe, depending on the pressure resistance and size of the filter.

[0047] The filtration step removes some or all of the viruses from the liquid. The means for removing aggregated viruses is not limited to the above filtration, and any known removal means can be used. For example, filtration using a filter with a finer pore size may be used.

[0048] The virus removal method according to this embodiment preferably includes a second step of adding ascorbic acid and / or a salt thereof to the virus-containing solution before the filtration step. The second step may be carried out before or after the first step, or may be carried out simultaneously with the first step. When the second step is carried out simultaneously with the first step, ascorbic acid and / or a salt thereof may be mixed with PQQ in advance and added simultaneously, or they may be added separately and simultaneously.

[0049] The virus removal method according to the present embodiment preferably includes a third step of adding one or more amino acids selected from acidic amino acids, neutral amino acids, and salts thereof to the virus-containing solution before the filtration step. The third step may be performed before or after the first step, or may be performed simultaneously with the first step. The third step may be performed before or after the second step, or may be performed simultaneously with the second step. When the third step is performed simultaneously with the first step and / or the second step, the amino acid may be mixed with PQQ and / or ascorbic acid in advance and added simultaneously, or may be added separately and simultaneously.

[0050] PQQ, ascorbic acid and / or its salt, and the above amino acid may be in the form of a powder or a solution when added to a solution containing viruses. The pH of the solution containing viruses when added can be weakly acidic to weakly basic. If the pH is strongly acidic or strongly basic, it becomes unsuitable for the manufacture of beverages or pharmaceuticals. The pH is preferably 2.5 to 8.5. The temperature of the solution containing viruses when added is not particularly limited, but 0°C to 45°C is preferable. If the temperature is below 0°C, the solution will freeze and will not be effective. Also, if the temperature is above 45°C, the solution will be in a heat-sterilized state, and will not be effective in removing viruses. Furthermore, it is not preferable for pharmaceuticals as it will deteriorate.

[0051] The time for addition and mixing can be appropriately determined depending on the purpose. Effectiveness can be achieved by contacting the virus for about 1 minute to 72 hours, preferably 5 minutes to 6 hours.

[0052] The definitions of each term, the content and content ratio of each component, etc. are the same as those explained for the virus aggregating agent.

[0053] [Virus Inhibitors] The virus inhibitor according to this embodiment includes pyrroloquinoline quinone, its derivatives and / or their salts, and ascorbic acid and / or its salts. The definitions of each term, such as virus, pyrroloquinoline quinone, its derivatives and / or their salts, and ascorbic acid and / or its salts, are the same as those explained for the virus aggregating agent.

[0054] The virus inhibitor according to the present embodiment has an effect of inhibiting virus activity, and can enhance the virus activity inhibitory effect in comparison with the case of using PQQ alone at the same concentration of PQQ. According to a preferred embodiment, for example, in a plaque assay performed in an example described later, the virus inhibition rate can be increased by 1.2 times or more, more preferably 1.3 times or more, even more preferably 1.4 times or more, and even more preferably 1.5 times or more in comparison with the case of using PQQ alone (comparative example).

[0055] The virus inhibitor of this embodiment preferably further comprises one or more amino acids selected from acidic amino acids, neutral amino acids, and salts thereof. By further comprising such amino acids in addition to PQQ, the virus activity inhibitory effect can be enhanced.

[0056] As the amino acid, isoleucine, leucine, methionine, phenylalanine, threonine (threonine), valine, tyrosine, cysteine, aspartic acid, asparagine, serine, glutamic acid, glutamine, proline, glycine, and alanine can be suitably used. Preferably, it is an acidic amino acid. Specifically, it is aspartic acid and glutamic acid. More preferably, it is glutamic acid. It is also preferable to contain a neutral amino acid, and it is particularly preferable to contain glycine. It may also contain a basic amino acid such as lysine, tryptophan, arginine, or histidine, but it is preferable not to contain it.

[0057] The salt of the amino acid is not particularly limited, and examples thereof include salts with alkali metals such as sodium and potassium, salts with alkaline earth metals such as calcium and magnesium, and salts with organic amines such as ammonium salts, aluminum salts, zinc salts, triethylamine, ethanolamine, morpholine, pyrrolidine, piperidine, piperazine, dicyclohexylamine, etc., for acidic groups such as carboxyl groups. Examples of salts with inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, and hydrobromic acid, salts with organic carboxylic acids such as acetic acid, citric acid, benzoic acid, maleic acid, fumaric acid, tartaric acid, succinic acid, tannic acid, butyric acid, hybenzic acid, pamoic acid, enanthic acid, decanoic acid, teoclic acid, salicylic acid, lactic acid, oxalic acid, mandelic acid, and malic acid, and salts with organic sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid, for basic groups such as amino groups.

[0058] The above amino acid may be mixed with PQQ or ascorbic acid and / or a salt thereof before being added to a virus-containing liquid to form a virus inhibitor, or PQQ, ascorbic acid and / or a salt thereof, and the above amino acid may be added to a virus-containing liquid, and then mixed in the liquid to form a virus inhibitor. Thus, the virus inhibitor according to this embodiment includes not only an embodiment in the form of a composition in which PQQ, ascorbic acid and / or a salt thereof, and the above amino acid are mixed, but also an embodiment in the form of a kit product in which each of them is manufactured and sold as a set.

[0059] The virus inhibitor according to the present embodiment may contain an effective amount of PQQ, and the amount is not limited. The effective amount of PQQ can be appropriately determined depending on the age, sex, weight, administration route, severity of symptoms, intended use, animal species, etc. of the subject to be ingested. For example, the amount is preferably 1 to 50% by mass, more preferably 10 to 50% by mass, and even more preferably 20 to 50% by mass, calculated as PQQ disodium trihydrate.

[0060] The content of ascorbic acid and / or a salt thereof in the virus inhibitor is preferably such that the mass ratio of the total content of pyrroloquinoline quinone, its derivatives and / or their salts to the total content of ascorbic acid and / or a salt thereof is 1:[1 to 100]. The mass ratio is more preferably 1:[1 to 20], and further preferably 1:[2 to 10].

[0061] The content of the amino acid in the virus inhibitor is preferably such that the mass ratio of the total content of pyrroloquinoline quinone, its derivatives and / or salts thereof: the total content of ascorbic acid and / or its salts: the total content of the one or more amino acids is 1:[1-100]:[1-100]. The mass ratio is more preferably 1:[1-20]:[1-20], and further preferably 1:[2-10]:[2-10].

[0062] The administration method of the virus inhibitor according to this embodiment is not limited to oral administration, and can also be by transdermal or injection, but oral administration is preferred. In the case of oral administration, powders, capsules, tablets, chewable tablets, orally disintegrating tablets (OD tablets), or drinks are preferred. In addition, it can be provided as a mouthwash to suppress virus proliferation in the oral cavity. It can also be mixed with food for use. PQQ is a functional food whose safety has already been confirmed, and it can be easily taken orally, so it has the advantage of being easy to implement medication compliance.

[0063] When the virus inhibitor according to the present embodiment is orally administered, the amount of PQQ is preferably 1 to 50 mg per day in terms of pyrroloquinoline quinone disodium, more preferably 5 to 40 mg, and even more preferably 10 to 30 mg. The number of times of administration per day may be once or multiple times.

[0064] The virus inhibitor according to the present embodiment may contain other components. The other components may be those that can be used as food. They can be appropriately selected according to the purpose, and are not particularly limited, but examples of the additives include excipients, binders, lubricants, disintegrants, solvents, stabilizers, solubilizers, antioxidants, colorants, flavorings, and sweeteners.

[0065] The excipient is not particularly limited, but examples thereof include sugars (white sugar, lactose, glucose, mannitol, etc.), starch (corn starch, etc.), crystalline cellulose, calcium phosphate, calcium sulfate, magnesium sulfate, etc.

[0066] The binder is not particularly limited, but examples thereof include pregelatinized starch, gelatin, gum tragacanth, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and polyvinyl alcohol.

[0067] The lubricant is not particularly limited, but examples thereof include magnesium stearate, talc, polyethylene glycol, silica, and hydrogenated vegetable oil.

[0068] The disintegrant is not particularly limited, but examples thereof include starch, crystalline cellulose, carboxymethylcellulose, agar, calcium citrate, calcium carbonate, sodium bicarbonate, dextrin, and the like.

[0069] The solvent is not particularly limited, but examples thereof include water, ethanol, glycerol, physiological saline, edible oils, etc. (soybean oil, fish oil, beeswax, lard, medium-chain fatty acid triglycerides), etc.

[0070] The stabilizer is not particularly limited, but examples thereof include benzoic acid, sodium benzoate, ethyl paraoxybenzoate, and propylene glycol.

[0071] The solubilizing agent is not particularly limited, but examples thereof include fumaric acid, succinic acid, and malic acid.

[0072] The antioxidant is not particularly limited, but examples thereof include ascorbic acid, tocopherol, sodium hydrogen sulfite, sodium thiosulfate, sodium pyrosulfite, and citric acid.

[0073] The coloring agents, flavoring agents, and sweetening agents include those that are generally permitted to be added in the pharmaceutical and food fields.

[0074] The subject of the virus inhibitor of this embodiment is mainly mammals. Suitable mammals include, for example, primates, livestock (e.g., sheep, dairy cows, horses, donkeys, pigs), laboratory animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g., cats, dogs) and captive wild animals (e.g., foxes, deer, dingoes). Preferably, humans and cats.

[0075] The virus inhibitor according to the present embodiment can be used by being contained in solid, semi-solid or liquid food and drink. Specific examples include health foods, functional foods, nutritional supplements, supplements, foods for specified health uses, foods for sick people, foods for the elderly, and retort foods. More specific examples of foods include confectionery (cookies, gummies, jellies, etc.), breads, rice, processed fish products, processed livestock products, noodles, soups, sauces, side dishes, and beverages (milk beverages, lactic acid bacteria beverages, soft drinks, vegetable beverages, powdered beverages, sports beverages, nutritional beverages, etc.). In addition to human foods, they can also be provided as pet foods and livestock feed. EXAMPLES

[0076] The present invention will be described in more detail below by way of examples, but the scope of the present invention is not limited in any way.

[0077] <Experimental Materials> PQQ was pyrroloquinoline quinone disodium salt (BioPQQ (product name, registered trademark) manufactured by Mitsubishi Gas Chemical). This BioPQQ is a trihydrate. The virus used was feline infectious peritonitis virus (FIPV: WSU 79-1146 strain), a type of feline coronavirus, obtained from ATCC (American Type Culture Collection). For preparation of virus stock solution and assay, CRFK cells, which are feline kidney-derived cells purchased from JCRB, were used. For other compounds and reagents, products manufactured by Fujifilm Wako Pure Chemical Corporation were used unless otherwise specified. The following were synthesized according to the literature. Reduced PQQ (RPQQ) was produced by reducing PQQ with VC (ascorbic acid) (Acta Crystallogr B Struct Sci Cryst Eng Mater. 2017 Jun 1;73(Pt 3):489-497.). Imidazolopyrroloquinoline (IPQ) was produced by the reaction of PQQ with glycine under aerobic conditions (J Am Chem Soc 1995, 117 (11), 3278-3279.). PQQ trimethyl ester (PQQ-TME) was synthesized by methylation of PQQ with dimethyl sulfate (Eur J Biochem 1980, 108 (1), 187-192.).

[0078] <Cell culture and preparation of virus stock solutions for experiments> (1)Cell culture The culture medium used was a mixture of 500 mL of E-MEM (WAKO), 50 mL of fetal bovine serum (Biowest), and 5 mL of penicillin streptomycin X100 (WAKO). CRFK cells were cultured in this E-MEM culture medium at 37°C under a 5% CO2 environment.

[0079] (2) Preparation of virus stock solution for experiments Violamo Culture Bottle 175cm 2 Add 25 mL of the E-MEM culture medium to the container and inject CRFK cells (80 × 10 5After culturing overnight, 25 mL of the culture medium was replaced. 1 mL of virus stock solution of FIPV:WSU 79-1146 strain was added to the culture medium. After culturing for 3 days, the bottle was placed in a -60°C freezer to freeze the culture medium. After 24 hours, the bottle was removed from the freezer, the culture medium was thawed, and the precipitate was removed by centrifugation (3000 rpm, 5 minutes). The culture medium from which the precipitate had been removed was filtered through a 0.22 μm filter to prepare a virus stock solution for experiments. The virus stock solution was stored at -60°C until use. The plaque-forming units of this virus stock solution were 2 × 10 5 The PFU / mL.

[0080] <Method for measuring the amount of virus removed> The amount of virus removed by the filter was measured. After mixing the virus with the virus flocculant of the present invention (PQQ or PQQ + various test substances), the mixture was filtered, and the amount of viral RNA in the filtrate that passed through was quantified by real-time PCR measurement. RNA was extracted from the virus using Cell Amp direct RNA prep kit (Takara Bio). The amount of RNA was quantified by RT-qPCR reaction using One Step (Takara Bio). (PCR conditions) The PCR conditions were as follows: Pattern 1 (reverse transcription reaction): 42°C for 5 minutes, 95°C for 10 seconds Pattern 2 (PCR reaction): Cycles 40, 95°C for 5 seconds, 60°C for 30 seconds The primers used had the following sequences:

[0081] [Table 1]

[0082] <Virus agglutination test>

[0083] (Comparative Example 1: Control) The prepared virus stock solution for experiments was diluted with PBS buffer (2 × 10 4 The amount of viral RNA measured by real-time PCR was set to 100%. The viral RNA reduction amount (hereinafter sometimes referred to as the viral reduction amount) was set to 0.

[0084] Example 1 The virus stock solution and PQQ were mixed in PBS buffer. The final virus concentration was 2 × 10 4 The experiment was performed so that the PFU / mL and PQQ concentration were 80 μg / mL. Filter filtration and real-time PCR measurement were performed in the same manner as in Comparative Example 1. The virus reduction amount was 10%. The results are shown in Table 2.

[0085] (Examples 2 to 7) As in Example 1, the experimental virus stock solution, PQQ, and various test substances (ascorbic acid, various amino acids, etc., listed in Table 2) were mixed using PBS buffer. The virus concentration was 2 × 10 4 The experiment was performed with PFU / mL, PQQ concentration of 80 μg / mL, and various additive concentrations of 400 μg / mL. Various test solutions were incubated at 37°C for 1 hour. After that, the test solutions were filtered using a 1 mL syringe and a membrane disk filter (cellulose acetate filter with a pore size of 0.20 μm: ADVANTEC DISMIC-13CP) to remove aggregated virus particles. The virus in the filtrate was quantified. The relative proportion of viral RNA was calculated by the following calculation: Viral RNA (relative ratio) = 2^(-(Ct value of control - Ct value of each sample)) From this, the amount of virus reduction was calculated by 100 × {1-virus RNA (relative ratio)}. The results are shown in Table 2.

[0086] [Table 2]

[0087] From Table 2, it can be seen that PQQ has a virus reduction effect, that is, a virus removal effect by filtration using a general filter. The virus reduction effect was improved by combined use with ascorbic acid, calcium chloride, etc. In particular, the virus reduction effect was dramatically improved by combined use of PQQ with ascorbic acid and an acidic amino acid (Examples 5 and 6).

[0088] From the above, it is believed that virus particles of a size that would pass through the pores of the filter were aggregated by PQQ and were unable to pass through the pores of the filter. This was confirmed by particle size measurement.

[0089] <Checking particle size distribution> (1) Experimental preparation Virus concentration (33-fold concentration from 50 mL to 1.5 mL) was performed as follows. CRFK cells were grown at 175 cm 2 The cells were cultured in two bottles at 37°C in a 5% CO2 environment until they reached confluence. The medium was removed, and 25 mL of medium and 800 μL of virus stock solution were placed in the bottles. The cells were cultured for three days. The bottles were frozen at -80°C, and the medium was thawed at room temperature. The cells were filtered through a 0.45 μm filter to obtain 50 mL of virus solution (2 × 10 5 A virus concentration of 1.5 PFU / mL was obtained. 50 mL of this virus solution was placed into four ultrafiltration membranes CFU100K. Centrifugation was performed at 3000 rpm for 30 minutes. 4 mL of virus concentrate was obtained. 16 mL of PBS buffer was added to this and placed into two ultrafiltration membranes CFU100K. Centrifugation was performed at 3000 rpm for 30 minutes. 2 mL of virus concentrate was obtained. 8 mL of PBS buffer was added to this and placed into one ultrafiltration membrane CFU100K. Centrifugation was performed at 3000 rpm for 30 minutes. 1.5 mL of virus concentrate was obtained. 200 μL was dispensed. This solution was used for particle size measurement.

[0090] (2) Measurement of particle size Each sample was fixed and inactivated with a 1% glutaraldehyde + 1% formaldehyde solution. Using a Horiba Nano Particle analyzer (SZ-100), the sample was diluted 100-fold with phosphate buffer (pH 7.4, GIBCO) and the particle size of each sample was measured. The results are shown in Table 3 and Figures 1 to 3. This shows that the virus particles aggregated and became gigantic when treated with PQQ.

[0091] [Table 3]

[0092] <Confirmation of virus aggregation effect by reduced PQQ and PQQ derivatives> In this study, reduced PQQ, and the PQQ derivatives imidazopyrroloquinoline (IPQ) and trimethyl ester PQQ were used. The PQQ derivative was dissolved in DMSO to 50 mM and diluted with PBS buffer to 100 μM. When ascorbic acid (VC) and glutamic acid were added, they were adjusted to 0.8 μg / μL each. The virus aggregation test described above was carried out in the same manner. The results are shown in Table 4. This confirmed that reduced PQQ and trimethyl ester PQQ have a greater virus-reducing effect, i.e., a greater virus-aggregating effect, than oxidized PQQ.

[0093] [Table 4]

[0094] <Plaque assay> (Culture medium used) Medium 1: Culture medium made by mixing 500 mL of E-MEM (WAKO) with 50 mL of fetal bovine serum (Biowest) and 5 mL of penicillin streptomycin X100 (WAKO). Medium 2: Medium 1 containing 1% carboxymethylcellulose

[0095] (Plaque assay (part 1)) 2 × 10 CRFK cells 5 1 mL of medium 1 was placed in a 24-well plate so that the number of cells / well was 100 PFU, and cultured overnight. 200 μL of a mixture was prepared using an experimental virus stock solution (virus amount 100 PFU) and the medium of each virus inhibitor (medium only in Comparative Example 3) in an amount that resulted in the final concentration shown in Table 5, and treated at 37°C for 1 hour. This mixture was contacted with the cells from which the medium had been removed for 1 hour. 1 mL of medium 2 was added to this. After 3 days, the medium was removed and the cells were fixed by treating with 10% paraformaldehyde PBS for 1 hour. The liquid was removed and washed with water. The cells were stained with crystal violet. The number of plaques was counted to measure the virus inhibition rate. The results are shown in Table 5. Plaques refer to holes that are formed when cells that have grown over the entire surface of the container are destroyed by virus infection. This number corresponds to the number of viruses.

[0096] (Plaque assay (part 2)) 2 × 10 CRFK cells 5 1 mL of medium 1 was placed in a 24-well plate so that the cells / well were cultured overnight. 200 μL of a mixture was prepared using an experimental virus stock solution (virus amount 100 PFU) and the medium of various virus inhibitors in an amount that would result in the final concentration shown in Table 6 (medium in Comparative Example 6), and treated at 37°C for 1 hour. This mixture was contacted with the cells from which the medium had been removed for 1 hour. The liquid was then removed and the cells were washed with medium. 1 mL of medium 2 was added to this. After 3 days, the medium was removed and the cells were fixed by treating with 10% paraformaldehyde PBS for 1 hour. The liquid was removed and the cells were washed with water. The cells were stained with crystal violet. The number of plaques was counted to measure the virus inhibition rate. The results are shown in Table 6.

[0097] [Table 5]

[0098] [Table 6]

Claims

1. A virus aggregating agent comprising pyrroloquinoline quinone, a derivative thereof and / or a salt thereof.

2. The virus aggregating agent according to claim 1 , further comprising ascorbic acid and / or a salt thereof.

3. The virus aggregating agent according to claim 2 , further comprising one or more amino acids selected from acidic amino acids, neutral amino acids and salts thereof.

4. The virus aggregating agent according to claim 3 , comprising an acidic amino acid, and wherein the acidic amino acid is glutamic acid.

5. The virus aggregating agent according to claim 3 , comprising a neutral amino acid, and including glycine as the neutral amino acid.

6. The virus aggregating agent according to claim 2, wherein the mass ratio of the total content of pyrroloquinoline quinone, its derivatives and / or salts thereof to the total content of ascorbic acid and / or its salts is 1:[1 to 100].

7. The virus aggregating agent according to claim 3, wherein the mass ratio of the total content of pyrroloquinoline quinone, its derivatives and / or their salts: the total content of ascorbic acid and / or its salts: the total content of the one or more amino acids is 1:[1 to 100]:[1 to 100].

8. The virus aggregating agent according to any one of claims 1 to 7, wherein the virus is a coronavirus.

9. The virus coagulant according to any one of claims 1 to 7, which is used as a pretreatment agent for removing viruses.

10. A virus inhibitor comprising pyrroloquinoline quinone, its derivatives and / or salts thereof, and ascorbic acid and / or its salts.

11. The virus inhibitor of claim 10, further comprising one or more amino acids selected from acidic amino acids, neutral amino acids and salts thereof.

12. The virus inhibitor according to claim 11, comprising said acidic amino acid, and wherein said acidic amino acid comprises glutamic acid.

13. The virus inhibitor according to claim 11 , comprising said neutral amino acid, and comprising glycine as said neutral amino acid.

14. The virus inhibitor according to claim 10, wherein the mass ratio of the total content of pyrroloquinoline quinone, its derivatives and / or salts thereof to the total content of ascorbic acid and / or its salts is 1:[1 to 100].

15. The virus inhibitor according to claim 11, wherein the mass ratio of the total content of pyrroloquinoline quinone, its derivatives and / or salts thereof: the total content of ascorbic acid and / or salts thereof: the total content of the one or more amino acids is 1:[1 to 100]:[1 to 100].

16. The virus inhibitor according to any one of claims 10 to 15, wherein the virus is a coronavirus.

17. A method for removing viruses, comprising a first step of adding pyrroloquinoline quinone, a derivative thereof and / or a salt thereof to a solution containing the viruses.

18. The virus removal method according to claim 17, further comprising, after the first step, a filtration step of filtering the solution through a filter having a pore size of 0.1 to 2 micrometers.

19. The method for removing viruses according to claim 18, further comprising a second step of adding ascorbic acid and / or a salt thereof to the solution prior to the filtration step.

20. The method for removing viruses according to claim 19, further comprising a third step of adding, before the filtration step, one or more amino acids selected from acidic amino acids, neutral amino acids, and salts thereof to the solution.

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