antiviral agents

Abstract

To provide an agent having antiviral action on a virus that can cause the novel coronavirus infection.SOLUTION: One embodiment of the present invention relates to an antiviral agent against the novel coronavirus (SARS-CoV-2) containing processed blue berries as an active ingredient. Another embodiment of the present invention relates to a food for preventing or treating a symptom or disorder in a subject having a SARS-CoV-2-induced viral infection, containing processed blue berries as an active ingredient.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an antiviral agent. In particular, one aspect of the present invention relates to an antiviral agent against the novel coronavirus, which contains a processed blueberry product as an active ingredient. [Background technology]

[0002] Since the beginning of 2020, an infectious disease caused by a novel coronavirus (hereinafter referred to as "SARS-CoV-2") (hereinafter referred to as "COVID-19") has spread worldwide. It is said that a certain percentage of COVID-19 patients develop severe symptoms. For this reason, saving the lives of severely ill COVID-19 patients has become a critical issue in the treatment of COVID-19 patients. The mortality rate for patients with severe pneumonia is high.

[0003] In Japan, remdesivir and favipiravir have been approved as antiviral drugs for COVID-19. However, there are situations where these approved drugs cannot be used.

[0004] In addition to SARS-CoV-2, various viruses such as influenza virus and severe acute respiratory syndrome coronavirus (hereinafter referred to as "SARS") have caused pandemics in recent years. It is also known that infection with viruses such as human T-cell leukemia virus (hereinafter referred to as "HTLV") and hepatitis C virus (hereinafter referred to as "HCV") can lead to the development of cancers such as adult T-cell leukemia and liver cancer.

[0005] In order to develop antiviral agents against various viral infections and cancer-causing viruses, active ingredients have been searched for by various means.

[0006] For example, Patent Document 1 describes a cell proliferation inhibitor against HTLV-1-infected cells or adult T-cell leukemia cells, which contains a processed blueberry leaf product as an active ingredient and is used to prevent the onset of diseases associated with HTLV-1 infection or to ameliorate or prevent the onset of adult T-cell leukemia.

[0007] Patent Document 2 describes an agent for suppressing hepatitis C virus production, which contains a composition comprising proanthocyanidins as an active ingredient. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent No. 4617418 [Patent Document 1] Patent No. 4892690 Summary of the Invention [Problem to be solved by the invention]

[0009] In Japan, remdesivir and favipiravir have been approved as antiviral drugs for COVID-19. Regarding the use of these approved drugs in COVID-19 treatment, the guidelines proposed by the Japanese Society of Acute Medicine and the Japanese Society of Intensive Care Medicine (Special Edition of the Japanese Sepsis Clinical Practice Guidelines 2020 (J-SSCG2020)) state that favipiravir is "weakly recommended" for mildly symptomatic patients, but is not currently "recommended" for moderately and severely symptomatic patients. Furthermore, remdesivir is not currently "recommended" for mildly symptomatic patients, but is "weakly recommended" for moderately and severely symptomatic patients. While these drugs are somewhat effective depending on the severity of the disease, their effectiveness is limited, and they cannot be a panacea for COVID-19. Therefore, new preventive or therapeutic measures for COVID-19 are needed.

[0010] Not only synthetic drugs that exhibit antiviral activity, such as remdesivir and favipiravir, but also natural physiologically active substances that exhibit antiviral activity, such as the drugs described in Patent Documents 1 and 2, are known. However, because SARS-CoV-2 is a novel virus, no drugs with antiviral activity against SARS-CoV-2 were known.

[0011] Therefore, the present invention aims to provide a drug having antiviral activity against SARS-CoV-2, which can cause COVID-19. [Means for solving the problem]

[0012] The present inventors have investigated various means for solving the above problems and have found that processed blueberries have an antiviral effect against SARS-CoV-2. Based on this finding, the present inventors have completed the present invention.

[0013] That is, the present invention includes the following aspects and embodiments. (1) An antiviral agent against the new coronavirus (SARS-CoV-2) that contains processed blueberries as an active ingredient. (2) The antiviral agent according to embodiment (1) for preventing or treating one or more viral infections selected from the group consisting of viral pneumonia, viral myocarditis, viral encephalitis, viral vasculitis, and viral conjunctivitis caused by the novel coronavirus (SARS-CoV-2). (3) The antiviral agent according to the above embodiment (1) or (2), wherein the processed blueberry product is a processed blueberry stem or leaf. (4) The antiviral agent according to any one of the above embodiments (1) to (3), wherein the processed blueberry product is one or more products or fractions selected from the group consisting of blueberry pulverization, blueberry juice, and solvent extract, and separated fractions thereof. (5) The antiviral agent according to any one of the above embodiments (1) to (4), wherein the antiviral effect of the processed blueberry product is due to at least one of the inhibition of viral growth and the inhibition of viral invasion into host cells. (6) Food containing processed blueberries as an active ingredient for the prevention or treatment of symptoms or disorders in subjects with a viral infection caused by the novel coronavirus (SARS-CoV-2). (7) The food according to the above embodiment (6), wherein the processed blueberry product is a processed blueberry stem or leaf. (8) The food product according to the above embodiment (6) or (7), wherein the processed blueberry product is one or more products or fractions selected from the group consisting of crushed blueberry, squeezed juice, solvent extract, and isolated fractions thereof. (9) The food according to any one of the above embodiments (6) to (8), wherein the processed blueberry product prevents or treats symptoms or disorders of viral infections through an antiviral effect resulting from at least one of the inhibition of viral proliferation and the inhibition of viral invasion into host cells. [Effects of the Invention]

[0014] The present invention makes it possible to provide drugs that have antiviral effects against SARS-CoV-2, which can cause COVID-19. [Brief explanation of the drawings]

[0015] [Figure 1] Figure 1 is a photograph of a host cell plate on which plaques formed after staining in Test II-1. In the figure, "Control" indicates the test result in which the same amount of DMSO was added instead of the sample, "Stem Extract" indicates the test result in which Fr. 7 of the blueberry stem extract obtained in Test I-1 was added, and "Leaf Extract" indicates the test result in which Fr. 7 of the blueberry leaf extract obtained in Test I-2 was added. [Figure 2]2 is a graph showing the relationship between the concentration of blueberry stem or leaf extract and the plaque reduction rate in Test II-2, where (a) shows the test results when blueberry stem extract was added, and (b) shows the test results when blueberry leaf extract was added. [Figure 3] 3 is a graph showing the relationship between blueberry stem or leaf extract concentration and 3CL protease activity in Test III-1. In the figure, A shows the test results when Fr. 7 of blueberry stem extract and leaf extract was added, and B shows the test results when Fr. 1 of blueberry stem extract and leaf extract was added. In A and B, (a) shows the test results when blueberry stem extract was added, and (b) shows the test results when blueberry leaf extract was added. The horizontal axis represents the logarithm of the extract concentration (log (μg / mL)), and the vertical axis represents protease activity (%). [Figure 4] 4 is a graph showing the relationship between the timing of addition of blueberry stem or leaf extract and the viral titer in Test III-2. In the graph, the horizontal axis represents the timing of addition of blueberry stem or leaf extract, and the vertical axis represents the logarithm of the viral titer (log(PFU / mL)). [Figure 5] 5 is a graph showing the relationship between the timing of addition of blueberry stem or leaf extract and the plaque reduction rate in Test III-2, where the horizontal axis represents the timing of addition of blueberry stem or leaf extract, and the vertical axis represents the plaque reduction rate (%). DETAILED DESCRIPTION OF THE INVENTION

[0016] The present inventors have found that processed blueberries have an antiviral effect against SARS-CoV-2. Therefore, one aspect of the present invention relates to an antiviral agent against SARS-CoV-2 that contains processed blueberries as an active ingredient.

[0017] In each embodiment of the present invention, the virus to which the antiviral agent is applied is the novel coronavirus (SARS-CoV-2). No effective treatment has yet been established for COVID-19, which is caused by SARS-CoV-2. The processed blueberry product, which is the active ingredient of the antiviral agent of this embodiment, has antiviral activity against SARS-CoV-2. Therefore, by applying the antiviral agent of this embodiment to SARS-CoV-2, antiviral effects such as growth inhibition, inactivation, and / or death of the virus can be exerted in vitro or in vivo. Furthermore, by administering the antiviral agent of this embodiment to a subject with COVID-19, a viral infection caused by SARS-CoV-2, such as a human patient with the viral infection, the symptoms or disorders of the viral infection can be prevented or treated via the antiviral activity of the processed blueberry product.

[0018] It has been found that the antiviral effect of the processed blueberry product, which is the active ingredient of the antiviral agent of this embodiment, is due to at least one of the following (i.e., either one or both): suppression of viral proliferation and suppression of viral invasion into host cells. The processed blueberry product can suppress viral proliferation, for example, by inhibiting the activity of 3CL protease, which is involved in processing the polyprotein translated from viral RNA. Furthermore, the processed blueberry product can strongly suppress viral invasion into host cells by being administered to a subject with COVID-19, a viral infection caused by SARS-CoV-2, such as a human patient with the viral infection, before or during infection of the host cells, or both.

[0019] In each embodiment of the present invention, viral infections caused by the viruses exemplified above include, but are not limited to, viral pneumonia, viral myocarditis, viral encephalitis, viral vasculitis, and viral conjunctivitis, particularly viral pneumonia. Viral pneumonia in subjects with viral pneumonia, e.g., human patients with viral pneumonia, is typically severe SARS-CoV-2 pneumonia, particularly severe SARS-CoV-2 pneumonia requiring mechanical ventilation. As used herein, "severe pneumonia" refers to pneumonia with symptoms such as respiratory failure. Also, as used herein, "mechanical ventilation" refers to mechanically ensuring breathing using a ventilator. In each embodiment of the present invention, administering the antiviral agent of this embodiment to a subject with COVID-19, a viral infection caused by SARS-CoV-2, e.g., a human patient with the viral infection, can prevent or treat symptoms or disorders of the viral infection via the antiviral effect of a processed blueberry product.

[0020] Symptoms or disorders of viral infections in subjects with COVID-19, a viral infection, e.g., human patients with the viral infection, include, but are not limited to, organ dysfunction, respiratory dysfunction (e.g., cough or dyspnea), fever, muscle pain, general fatigue, sepsis, septic shock, and multiple organ failure (e.g., organ failure of the lungs, kidneys, liver, heart, blood vessels, or cranial nerves) in the case of viral pneumonia; myocarditis in the case of viral myocarditis; encephalitis in the case of viral encephalitis; vasculitis in the case of viral vasculitis; and conjunctivitis in the case of viral conjunctivitis. In each embodiment of the present invention, by administering the antiviral agent of this embodiment to a subject with COVID-19, a viral infection caused by SARS-CoV-2, e.g., a human patient with the viral infection, the antiviral effect of the processed blueberry product can be used to prevent or treat symptoms or disorders of the viral infection.

[0021] In each aspect of the present invention, the antiviral activity of a blueberry processed product can be determined, for example, by culturing a virus and its host cell in the presence of the blueberry processed product and evaluating the inhibition of virus growth (e.g., inhibition of plaque formation), inactivation, and / or killing of the virus. Alternatively, the activity can be determined by administering the blueberry processed product to a subject, such as a human patient, infected with a virus and measuring the virus concentration in the subject's body.

[0022] In each embodiment of the present invention, the preventive or therapeutic effect of a viral infection on symptoms or disorders in a subject with a viral infection, such as a human patient with a viral infection, can be determined, for example, by administering an antiviral agent of this embodiment to a subject with a viral infection and evaluating the symptoms or disorders of the viral infection in the subject using, for example, the treatment period (e.g., the period during which mechanical ventilation was required), clinical condition, laboratory tests (e.g., hematology tests and blood biochemistry tests), vital signs (e.g., blood pressure and heart rate), and changes in inflammatory cytokines (e.g., IL-1β, IL-6, IL-8, IL-10, TNF-α, IFN-γ, autotaxin, FDP, D-dimer, tPA, PIC, and PTX3) as indicators. In addition, the preventive or therapeutic effect of a viral infection on symptoms or disorders in a subject with a viral infection, such as a human patient with a viral infection, can also be determined by evaluating the symptoms or disorders using, for example, the quantitative results of the virus using a gene detection method such as PCR or an immunological method as an indicator. Furthermore, in each embodiment of the present invention, the occurrence of undesirable serious side effects (e.g., circulatory failure (shock) due to an excessive decrease in blood pressure) when the antiviral agent of this embodiment is administered to a subject with a viral infection, for example, a human patient with a viral infection, can be determined, for example, by observing circulatory dynamics such as blood pressure.

[0023] As used herein, "prevention" means substantially preventing the occurrence (development or manifestation) of a symptom or disorder in a subject with a viral infection, e.g., a human patient with a viral infection. Also, as used herein, "treatment" means suppressing (e.g., suppressing progression of), alleviating, repairing, and / or curing a symptom or disorder that has occurred (development or manifestation) in a subject with a viral infection, e.g., a human patient with a viral infection.

[0024] Blueberries are deciduous or evergreen, shrub-like or semi-tree-like fruit trees native to America, classified as part of the Ericaceae family, Vaccinium genus, Cyanococcus section (Vander Kloet, 1988; Tamada, 1996). Blueberries consist of approximately six species, but the three most important in horticulture are (1) highbush blueberry (Vaccinium corymbosum L.), (2) rabbiteye blueberry (V. virgatum Aiton), and (3) lowbush blueberry (V. angustifolium Michaux and V. myrtilloides Aition). These blueberry species were developed from wild species by the United States Department of Agriculture or state universities beginning in the early 20th century. The direction of breeding was to aim for ease of cultivation and the production of large, sweet, high-quality blueberries. Today, blueberries are cultivated all over the world. In recent years, uses for parts of the plant other than the fruit (for example, food uses) have also been developed, such as using blueberry leaves as tea.

[0025] In each aspect of the present invention, the type, origin, and place of origin of blueberries used as an active ingredient are not particularly limited. Blueberries of any type, origin, and place of origin, including the species exemplified above, can be used as an active ingredient.

[0026] In each embodiment of the present invention, the processed blueberry used as an active ingredient may be a processed product of any part of the plant, but is typically a processed product of any part other than the fruit, such as a processed product of the stem, leaf, flower, or root, and particularly a processed product of the stem or leaf. The blueberry plant parts exemplified above may be used alone or in combination of one or more. As shown in the following examples, processed blueberries have a high antiviral effect against SARS-CoV-2. Therefore, by administering the antiviral agent of this embodiment to a subject with COVID-19, a viral infection caused by SARS-CoV-2, such as a human patient with the viral infection, the antiviral effect of the processed blueberry product can be used to prevent or treat symptoms or disorders of the viral infection.

[0027] In each embodiment of the present invention, blueberries used as an active ingredient may be used in their original form, but are typically used in the form of a processed product. In each embodiment of the present invention, "processed product" refers to a product or fraction that has been processed or separated by physical or chemical means. Methods for obtaining processed products include, but are not limited to, crushing, squeezing, drying, solvent extraction, filtration, centrifugation, adsorption, recrystallization, distillation, and various types of chromatography. Each of the above methods may be combined and / or repeated multiple times under the same or different conditions, as desired.

[0028] The pulverization can be carried out using a pulverizer such as a mill or blender commonly used in the art. For example, pulverized blueberries can be obtained by pulverizing blueberry parts, preferably stems or leaves, either as they are or after drying. The pulverized product may be dried if desired.

[0029] Drying can be carried out by any means and under any conditions as long as the antiviral activity of the processed blueberry product is not impaired. Drying can be carried out by means of, for example, vacuum freeze drying, hot air drying, far-infrared drying, reduced-pressure drying, microwave reduced-pressure drying, or superheated steam drying. Because this method results in minimal changes in the components of the processed product, it is preferable to dry blueberries or their processed product (e.g., pulverized product) by vacuum freeze drying.

[0030] Solvent extraction can be carried out using, but is not limited to, water or an organic solvent such as a lower alcohol (e.g., an alcohol having 1 to 6 carbon atoms, such as methanol, ethanol, or 2-propanol (isopropyl alcohol)), a higher alcohol (e.g., an alcohol having 7 or more carbon atoms, such as 1-heptanol or 1-octanol), acetone, acetonitrile, diethyl ether, or ethyl acetate. The solvent used for solvent extraction of blueberries or a processed product thereof (e.g., a crushed product or a squeezed juice) is preferably water or a lower alcohol, more preferably water. Furthermore, solvent extraction may be carried out at room temperature or under heated conditions. Particularly preferably, a blueberry solvent extract can be obtained by hot water extraction of blueberries or a processed product thereof (e.g., a crushed product or a squeezed juice). The solvent extract may be dried, if desired.

[0031] Chromatography can be carried out using various chromatographic supports commonly used in the art, such as adsorption, normal or reverse phase partition, or gel filtration.

[0032] The processed blueberry product is preferably one or more products or fractions selected from the group consisting of crushed blueberry plant parts (e.g., stems or leaves) exemplified above, squeezed juice, and solvent extracts, and separated fractions thereof, more preferably a solvent extract of the crushed blueberry plant parts or a separated fraction of the solvent extract, and even more preferably a hot water extract of the crushed blueberry plant parts (e.g., stems or leaves) exemplified above, separated by chromatography using Sephadex. (registered trademark) This fraction is applied to LH-20 column chromatography and eluted with an eluent of distilled water:acetone = 3:2. By administering the antiviral agent of this embodiment, which contains the above-exemplified processed product of blueberries as an active ingredient, to a subject with COVID-19, a viral infection caused by SARS-CoV-2, such as a human patient with said viral infection, it is possible to obtain a preventive or therapeutic effect on the symptoms or disorders of said viral infection via the antiviral action of the processed product of blueberries.

[0033] Due to the above characteristics, processed blueberries can exhibit antiviral effects such as inhibiting viral growth, inactivating, and / or killing viruses, and through the antiviral effects, can prevent or treat symptoms or disorders of viral infections in subjects with viral infections, such as human patients with viral infections.

[0034] As explained above, processed blueberries can exhibit antiviral effects such as inhibiting viral growth, inactivating, and / or killing viruses. Therefore, another aspect of the present invention relates to a pharmaceutical composition for preventing or treating symptoms or disorders in a subject with a viral infection, comprising a processed blueberry composition as an active ingredient. The pharmaceutical composition of this aspect can prevent or treat symptoms or disorders in a subject with a viral infection through antiviral effects such as inhibiting viral growth, inactivating, and / or killing viruses.

[0035] As explained above, the antiviral effect of the processed blueberry product, which is the active ingredient of the pharmaceutical of this embodiment, is due to at least one of the inhibition of viral growth and the inhibition of viral invasion into host cells (i.e., either or both). Therefore, the processed blueberry product, which is the active ingredient of the pharmaceutical of this embodiment, can prevent or treat symptoms or disorders of viral infection in a subject with a viral infection through the antiviral effect due to at least one of the inhibition of viral growth and the inhibition of viral invasion into host cells (i.e., either or both).

[0036] In the antiviral agent and medicament of this embodiment, the processed blueberry product used as the active ingredient may be used alone or in combination with one or more pharmaceutically acceptable ingredients. The antiviral agent and medicament of this embodiment can be formulated into various dosage forms commonly used in the art depending on the desired administration method. Therefore, the antiviral agent and medicament of this embodiment can also be provided in the form of a pharmaceutical composition containing the processed blueberry product and one or more pharmaceutically acceptable carriers. In this embodiment, the pharmaceutical composition may contain, in addition to the above ingredients, one or more pharmaceutically acceptable additives such as one or more pharmaceutically acceptable vehicles (e.g., solvents such as sterile water or solutions such as physiological saline), excipients, binders, vehicles, solubilizers, preservatives, stabilizers, disintegrants, disintegration inhibitors, bulking agents, lubricants, surfactants, emulsifiers, oily liquids (e.g., vegetable oils), suspending agents, buffers, soothing agents, antioxidants, sweeteners, and flavoring agents.

[0037] The dosage form of the antiviral agent and medicament of this embodiment is not particularly limited, and may be a preparation for use in parenteral administration, a preparation for use in transmucosal (e.g., nasal, sublingual, or oral mucosal), transdermal, transanal (enema), or transvaginal administration, or a preparation for use in oral administration. Furthermore, the dosage form of the antiviral agent and medicament of this embodiment may be a preparation in a unit dose form or a preparation in a multiple dose form. Examples of preparations for use in parenteral administration include injections such as sterile solutions or suspensions with water or other pharmaceutically acceptable liquids. Additives that can be mixed into injections include, but are not limited to, vehicles such as isotonic solutions containing physiological saline, glucose, or other auxiliary agents (e.g., D-sorbitol, D-mannitol, or sodium chloride), solubilizers such as alcohols (e.g., ethanol or benzyl alcohol), esters (e.g., benzyl benzoate), and polyalcohols (e.g., propylene glycol or polyethylene glycol), nonionic surfactants such as polysorbate 80 or polyoxyethylene hydrogenated castor oil, oily solutions such as sesame oil or soybean oil, buffers such as phosphate buffer or sodium acetate buffer, soothing agents such as benzalkonium chloride or procaine hydrochloride, stabilizers such as human serum albumin or polyethylene glycol, preservatives, and antioxidants. Prepared injections are usually filled into appropriate containers (e.g., vials or ampoules) and stored under appropriate conditions until use.

[0038] Formulations for oral administration include, but are not limited to, tablets, pills, powders, capsules, soft capsules, microcapsules, elixirs, liquids, syrups, slurries, suspensions, etc. Tablets may be formulated as sugar-coated tablets or sugar-coated tablets with a dissolving film, gelatin-coated tablets, enteric-coated tablets, orally disintegrating tablets (OD tablets), or film-coated tablets, or may be formulated as double tablets or multi-layer tablets, as desired.

[0039] Ingredients that can be mixed into tablets or capsules include, but are not limited to, binders such as water, ethanol, propanol, simple syrup, glucose solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, gelatin, corn starch, tragacanth gum, or gum arabic; excipients such as crystalline cellulose, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, or silicic acid; dry starch, sodium alginate, agar powder, laminaran powder, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, and the like. Examples of suitable carriers include disintegrants such as esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, or lactose; disintegration inhibitors such as sucrose, stearic cocoa butter, or hydrogenated oils; absorption promoters such as quaternary ammonium salts or sodium lauryl sulfate; moisturizing agents such as glycerin or starch; adsorbents such as starch, lactose, kaolin, bentonite, or colloidal silicic acid; lubricants such as purified talc, stearates (e.g., magnesium stearate), boric acid powder, or polyethylene glycol; sweeteners such as sucrose, lactose, or saccharin; and flavoring agents such as peppermint, sucrose oil, or cherry. When the formulation is a capsule, it may further contain a liquid carrier such as an oil or fat.

[0040] The antiviral agent and medicament of this embodiment can also be used in combination with one or more other pharmaceutical agents. In this case, the antiviral agent and medicament of this embodiment may be provided in the form of a single pharmaceutical agent containing a processed blueberry product and one or more other pharmaceutical agents, or may be provided in the form of a pharmaceutical combination or kit containing multiple preparations in which a processed blueberry product and one or more other pharmaceutical agents are separately formulated. In the case of a pharmaceutical combination or kit, the respective preparations can be administered simultaneously or separately (for example, consecutively).

[0041] Similar to the antiviral agent of one aspect of the present invention described above, the medicament of this aspect can be used to prevent or treat symptoms or disorders of a viral infection in a subject with a viral infection, for example, a human patient with a viral infection, through the antiviral activity of a processed blueberry product. In the medicament of this aspect, the viral infection is, for example, viral pneumonia, viral myocarditis, viral encephalitis, viral vasculitis, or viral conjunctivitis. In one embodiment, when the medicament of this aspect is applied to a subject with viral pneumonia, for example, a human patient with viral pneumonia, the medicament of this aspect is preferably used to prevent or treat organ damage, respiratory damage (e.g., cough or dyspnea), fever, muscle pain, general fatigue, sepsis, septic shock, or multiple organ failure (e.g., organ failure of the lungs, kidneys, liver, heart, blood vessels, or cranial nerves) in the subject or human patient, and more preferably used to prevent or treat organ damage in the subject or human patient. In another embodiment, when the medicament of this aspect is applied to a subject with viral myocarditis, for example, a human patient with viral myocarditis, the medicament of this aspect is preferably used to prevent or treat myocarditis in the subject or human patient. In another embodiment, when the medicament of this aspect is applied to a subject with viral encephalitis, for example, a human patient with viral encephalitis, the medicament of this aspect is preferably used to prevent or treat encephalitis in the subject or human patient. In another embodiment, when the medicament of this aspect is applied to a subject with viral vasculitis, for example, a human patient with viral vasculitis, the medicament of this aspect is preferably used to prevent or treat vasculitis in the subject or human patient. In another embodiment, when the medicament of this aspect is applied to a subject with viral conjunctivitis, for example, a human patient with viral conjunctivitis, the medicament of this aspect is preferably used to prevent or treat conjunctivitis in the subject or human patient.By using the pharmaceutical of this embodiment for the prevention or treatment of the symptoms or disorders of the viral infection exemplified above in a subject with a viral infection, for example, a human patient with a viral infection, the symptoms or disorders of the viral infection can be prevented or treated via the antiviral action of the processed blueberry product.

[0042] The processed blueberry product used as the active ingredient of the antiviral agent and medicament of this embodiment is derived from a blueberry plant. Therefore, the processed blueberry product is safe and low-toxic. Therefore, the antiviral agent and medicament of this embodiment can be applied to various subjects requiring prevention or treatment of symptoms or disorders of viral infections. The subject is preferably a human or non-human mammalian subject or patient (e.g., a warm-blooded animal such as a pig, dog, cow, rat, mouse, guinea pig, rabbit, chicken, sheep, cat, monkey, hamadryas baboon, or chimpanzee), and more preferably a human patient. By administering the antiviral agent and medicament of this embodiment to the subject, symptoms or disorders of viral infections in the subject can be prevented or treated via the antiviral effect of the processed blueberry product.

[0043] When the antiviral agent and medicament of this embodiment are administered to a subject, particularly a human patient, the exact dosage and administration method should ultimately be determined by the attending physician, taking into account many factors, such as the subject's age and sex, the symptoms to be prevented or treated, the exact state (e.g., severity) of the disease and / or disorder, and the route of administration. Therefore, in the antiviral agent and medicament of this embodiment, the processed product of blueberries, which is the active ingredient, is administered to the subject at a therapeutically effective dosage and administration method (e.g., dosage amount and administration route). For example, when the antiviral agent and medicament of this embodiment are administered to a subject, particularly a human patient, the dosage of the processed product of blueberries used as the active ingredient is typically in the range of 1 to 1,000 mg / kg body weight / day, for example, 10 to 100 mg / kg body weight / day.

[0044] The antiviral agent and medicament of this embodiment may be administered by any administration route. For example, the antiviral agent and medicament of this embodiment are preferably administered by routes such as intravenous administration, enema administration, subcutaneous administration, intramuscular administration, oral administration, or intraperitoneal administration, and more preferably orally.

[0045] As explained above, the processed blueberry product, which is the active ingredient of the antiviral agent and medicament of this embodiment, can be administered to a subject, for example, a human patient with COVID-19, a viral infection caused by SARS-CoV-2, before or during infection, or both, to strongly suppress viral entry into host cells. Therefore, the antiviral agent and medicament of this embodiment are preferably administered to a subject before or during viral infection, or both. By administering the antiviral agent and medicament of this embodiment at the times exemplified above, symptoms or disorders of viral infection in a subject can be more effectively prevented or treated.

[0046] Through its antiviral activity, such as inhibiting viral growth, inactivating, and / or killing viruses, processed blueberries can be used to prevent or treat symptoms or disorders of viral infections in subjects with viral infections, for example, human patients with viral infections. Therefore, another aspect of the present invention relates to a preventive or therapeutic agent for viral infections, comprising processed blueberries as an active ingredient. The preventive or therapeutic agent of this aspect has the same characteristics as the antiviral agent and medicament of this aspect described above. Furthermore, the preventive or therapeutic agent of this aspect can be used in the same dosage and administration as the antiviral agent and medicament of this aspect described above. Through the antiviral activity of processed blueberries, the preventive or therapeutic agent of this aspect can be used to prevent or treat symptoms or disorders of viral infections in subjects with viral infections, for example, human patients with viral infections. In the preventive or therapeutic agent of this aspect, the viral infection is, for example, viral pneumonia, viral myocarditis, viral encephalitis, viral vasculitis, or viral conjunctivitis. In the prophylactic or therapeutic agent of this embodiment, the symptoms or disorders of viral infections include, for example, in the case of viral pneumonia, organ dysfunction, respiratory dysfunction (e.g., cough or dyspnea), fever, muscle pain, general fatigue, sepsis, septic shock, or multiple organ failure (e.g., organ failure of the lungs, kidneys, liver, heart, blood vessels, or cranial nerves), particularly organ dysfunction; in the case of viral myocarditis, myocarditis; in the case of viral encephalitis, encephalitis; in the case of viral vasculitis, vasculitis; and in the case of viral conjunctivitis, conjunctivitis. By administering an effective amount of a processed blueberry product to a subject with a viral infection, for example, a human patient with a viral infection, the above-mentioned symptoms or disorders of viral infections can be prevented or treated via the antiviral effect.

[0047] Another aspect of the present invention is a method for preventing or treating symptoms or disorders caused by a viral infection, comprising administering an effective amount of a processed blueberry product to a subject with a viral infection, such as a human patient, to exert an antiviral effect, such as inhibiting proliferation, inactivating, and / or killing the virus. The processed blueberry product administered in the method of this aspect has similar characteristics to the active ingredients of the antiviral agent and medicament of this aspect described above. Furthermore, the method of this aspect can be carried out in the same manner and dosage as the antiviral agent and medicament of this aspect described above. In the method of this aspect, the viral infection is, for example, viral pneumonia, viral myocarditis, viral encephalitis, viral vasculitis, or viral conjunctivitis. In the method of this embodiment, the symptoms or disorders of viral infections include, for example, in the case of viral pneumonia, organ dysfunction, respiratory dysfunction (e.g., cough or difficulty breathing), fever, muscle pain, general fatigue, sepsis, septic shock, or multiple organ failure (e.g., organ failure of the lungs, kidneys, liver, heart, blood vessels, or cranial nerves), particularly organ dysfunction; in the case of viral myocarditis, myocarditis; in the case of viral encephalitis, encephalitis; in the case of viral vasculitis, vasculitis; and in the case of viral conjunctivitis, conjunctivitis. By administering an effective amount of a processed blueberry product to a subject with a viral infection, for example, a human patient with a viral infection, the above-mentioned symptoms or disorders of viral infections can be prevented or treated via the antiviral effect.

[0048] Another aspect of the present invention is a processed product of blueberries for use in preventing or treating symptoms or disorders of a viral infection in a subject with a viral infection, for example, a human patient with a viral infection, through antiviral effects such as virus growth inhibition, inactivation, and / or killing. Yet another aspect of the present invention is use of a processed product of blueberries in the manufacture of a medicament for preventing or treating symptoms or disorders of a viral infection in a subject with a viral infection, for example, a human patient with a viral infection, through antiviral effects such as virus growth inhibition, inactivation, and / or killing. Yet another aspect of the present invention is use of a processed product of blueberries for preventing or treating symptoms or disorders of a viral infection in a subject with a viral infection, for example, a human patient with a viral infection, through antiviral effects such as virus growth inhibition, inactivation, and / or killing. The compound, etc. of this aspect has similar characteristics to the active ingredients of the antiviral agent and medicament of this aspect described above. Furthermore, the compound, etc. of this aspect can be used in the same dosage and administration as the antiviral agent and medicament of this aspect described above. In the compound etc. or use of this embodiment, the viral infection is, for example, viral pneumonia, viral myocarditis, viral encephalitis, viral vasculitis, or viral conjunctivitis. In the compound etc. or use of this embodiment, the symptoms or disorders of viral infection are, for example, in the case of viral pneumonia, organ dysfunction, respiratory dysfunction (e.g., cough or dyspnea), fever, muscle pain, general fatigue, sepsis, septic shock, or multiple organ failure (e.g., organ failure of the lungs, kidneys, liver, heart, blood vessels, or cranial nerves), particularly organ dysfunction; in the case of viral myocarditis, myocarditis; in the case of viral encephalitis, encephalitis; in the case of viral vasculitis, vasculitis; and in the case of viral conjunctivitis, conjunctivitis. By administering an effective amount of a processed blueberry product to a subject with a viral infection, for example, a human patient with a viral infection, the symptoms or disorders of the viral infection exemplified above can be prevented or treated via the antiviral effect.

[0049] Because processed blueberries have been used in food applications, they have been established as safe and low-toxic. Therefore, another aspect of the present invention relates to a food product containing processed blueberries as an active ingredient for preventing or treating symptoms or disorders in a subject with the above-exemplified viral infections, particularly viral infections caused by SARS-CoV-2. The food product of this aspect can prevent or treat symptoms or disorders in a subject with the above-exemplified viral infections through antiviral effects such as viral growth inhibition, inactivation, and / or death, without substantially affecting the health of the subject consuming the food product.

[0050] As explained above, the antiviral effect of the processed blueberry product, which is an active ingredient of the food product of this embodiment, is due to at least one of the inhibition of viral growth and the inhibition of viral invasion into host cells (i.e., either or both). Therefore, the processed blueberry product, which is an active ingredient of the food product of this embodiment, can prevent or treat symptoms or disorders of viral infection in a subject with a viral infection through the antiviral effect due to at least one of the inhibition of viral growth and the inhibition of viral invasion into host cells (i.e., either or both).

[0051] In the food product of this embodiment, the processed blueberry product used as an active ingredient may be used alone or in combination with one or more edibly acceptable ingredients. The food product of this embodiment can be formulated into various dosage forms commonly used in the art depending on the desired method of ingestion. Therefore, the food product of this embodiment can also be provided in the form of a food composition containing the processed blueberry product and one or more edibly acceptable ingredients. In this embodiment, the one or more edibly acceptable ingredients may include preservatives, stabilizers, dispersants, bulking agents, surfactants, oily liquids, buffers, antioxidants, sweeteners, flavorings, colorings, pigments, etc.

[0052] The food product of this embodiment may be used as a food product as it is, or may be mixed with other foods or food components, i.e., used as a food ingredient.

[0053] The food of this embodiment can be used, for example, as a dietary supplement, functional food, or food for specified health uses. The food of this embodiment may take the form of, for example, a regular food or beverage, or a dietary supplement such as a supplement. Examples of dietary supplement forms include tablets, pills, powders, capsules, soft capsules, microcapsules, elixirs, liquids, syrups, slurries, and suspensions. Tablets may be formulated as sugar-coated or dissolvable-coated tablets, gelatin-coated tablets, enteric-coated tablets, orally disintegrating tablets (OD tablets), or film-coated tablets, or as double- or multi-layered tablets, as desired. The food of this embodiment can be formulated using the same ingredients as those of the pharmaceutical of one embodiment of the present invention described above.

[0054] When a subject, particularly a human, ingests the food product of this embodiment, the processed blueberry product as an active ingredient is ingested by the subject in a manner and dosage (e.g., intake amount and intake route) that is effective and safe for food. For example, when a subject, particularly a human, ingests the food product of this embodiment, the intake amount of the processed blueberry product used as an active ingredient is usually in the range of 1 to 1000 mg / kg body weight / day, for example, in the range of 10 to 100 mg / kg body weight / day.

[0055] As explained above, the processed blueberry product, which is the active ingredient of the food product of this embodiment, can be ingested by a subject, for example, a human patient with COVID-19, a viral infection caused by SARS-CoV-2, before or during infection, or both, to strongly suppress viral entry into host cells. Therefore, the food product of this embodiment is preferably ingested by a subject before or during viral infection, or both. Ingesting the food product of this embodiment at the times exemplified above can more effectively prevent or treat symptoms or disorders of viral infection in a subject. [Example]

[0056] The present invention will be described in more detail below using examples, although the technical scope of the present invention is not limited to these examples.

[0057] <Test I: Preparation of processed blueberries> [I-1: Fractionation of blueberry stem extract] An appropriate amount of water was added to the stems of blueberries (rabbiteye blueberry variety) based on their weight, and hot water extraction was performed at 90-95°C. The hot water extract was freeze-dried. 50.00 g of the freeze-dried hot water extract was then added to a 5000 cm 3 Weigh out 3000 cm of distilled water into an Erlenmeyer flask. 3 The mixture was sonicated at 50°C for 30 minutes to dissolve the extract. The temperature was then returned to room temperature, and the extract was filtered by suction using an aspirator. The filtrate was filtered through Sephadex LH-20 550 cm 3 The resulting fraction was designated as Fr. 0 (flow-through). Then, the solution was passed through a biocolumn (inner diameter 50 mm, length 500 mm) packed with distilled water at 1500 cm 3 , distilled water:methanol = 4:1 1000 cm 3 , distilled water:methanol = 3:2 1000 cm 3 , distilled water:methanol = 2:3 1000 cm 3 , distilled water:methanol = 1:4 1000 cm 3, methanol 1000 cm 3 , distilled water:acetone=3:2 1000 cm 3 , distilled water:acetone = 2:3 1000 cm 3 , and acetone 1000 cm 3 The eluate was passed through the biocolumn in this order to obtain fractions Fr. 1 to 9. The fractions obtained were lyophilized, either as they were if the eluate was only water, or after removing the organic solvent in an evaporator. After lyophilization, the yield of each fraction was calculated. The results are shown in Table 1 below.

[0058] [Table 1]

[0059] [I-2: Fractionation of blueberry leaf extract] An appropriate amount of water was added to blueberry (rabbiteye blueberry) leaves based on their weight, and hot water extraction was performed at 90-95°C. The hot water extract was freeze-dried. 20.41 g of the freeze-dried hot water extract was added to a 3000 cm 3 Weigh out 1200 cm of distilled water into an Erlenmeyer flask. 3 The mixture was sonicated at 50°C for 30 minutes to dissolve the extract. The temperature was then returned to room temperature, and the extract was filtered by suction using an aspirator. The filtrate was filtered through Sephadex LH-20 220 cm 3 The resulting fraction was designated as Fr. 0 (pass-through). Then, 600 cm of distilled water was added. 3 , distilled water:methanol = 4:1 500 cm 3 , distilled water:methanol = 3:2 500 cm 3 , distilled water:methanol = 2:3 500 cm 3 , distilled water:methanol = 1:4 500 cm 3 , methanol 500 cm 3 , distilled water:acetone=3:2 500 cm 3 , distilled water:acetone = 2:3 500 cm 3, and acetone 500 cm 3 The eluate was passed through the biocolumn in this order to obtain fractions Fr. 1 to 9. The fractions obtained were lyophilized, either as they were if the eluate contained only water, or after removing the organic solvent in an evaporator. After lyophilization, the yield of each fraction was calculated. The results are shown in Table 2 below.

[0060] [Table 2]

[0061] <Test II: Antiviral effect of processed blueberries> [II-1: Plaque formation inhibition test (1)] The novel coronavirus (SARS-CoV-2 / Hu / DP / Kng / 19-027, LC528233, provided by the Kanagawa Prefectural Institute of Public Health) was used as the inoculation virus, and Vero cells (Vero-TMPRSS2) were used as the host cells. The host cells were seeded and cultured in a 12-well plate containing MEM medium. The culture medium was removed from the cultured 12-well plate, and 500 μL of fresh medium containing a 50 μg / mL DMSO solution of the sample was added. The host cells were cultured in a carbon dioxide incubator at 5% CO2 and 37°C for 5 hours. The culture medium was removed, and 1 × 10 4500 μL of the inoculum, prepared by diluting the PFU virus stock solution 10-fold or 100-fold, was added to each well. For the untreated control group, the same volume of fresh medium was added instead of the inoculum. The virus was allowed to adsorb to the host cells for 2 hours. The inoculum was then removed. Fresh medium containing a 50 μg / mL DMSO solution of the sample and carboxymethylcellulose (CMC) was added to each well, 1 mL per well, and layered onto the host cells. The host cells were then cultured in a carbon dioxide incubator at 5% CO2 and 37°C for 3 days. The culture medium was removed, and the host cells were fixed by adding methanol and allowing to stand for 5 minutes. Crystal violet solution (30% ethanol solution containing 2.5% crystal violet) was then added and stained for 20 minutes. The host cells were then observed under an optical microscope to count the number of plaques. The percentage of plaques formed in the sample-treated group compared to the control group was calculated as the plaque reduction rate (%). A photograph of the stained host cell plate with plaques is shown in Figure 1. In the figure, "Control" shows the test results when the same amount of DMSO was added instead of the sample, "Stem Extract" shows the test results when Fr. 7 of the stem extract obtained in Test I-1 was added, and "Leaf Extract" shows the test results when Fr. 7 of the leaf extract obtained in Test I-2 was added.

[0062] As shown in Figure 1, the addition of blueberry stem extract F. 7 or leaf extract F. 7 significantly inhibited plaque formation by SARS-CoV-2. For example, the 100-fold diluted inoculum virus solution (1 × 10 2 In the test using PFU, the number of plaques formed in the control was over 66 (smear), whereas the number of plaques formed in the addition of Fr. 7 of stem extract was less than 1 (plaque reduction rate of over 98.5%), and the number of plaques formed in the addition of Fr. 7 of leaf extract was 1 (plaque reduction rate of over 98.5%).

[0063] [II-2: Plaque formation inhibition test (2)] A plaque formation inhibition test was conducted using the same procedure as in Test II-2, in which a sample solution prepared to a predetermined concentration (0.08, 0.4, 2, 10, or 50 μg / mL) was added. Figure 2 shows the relationship between the concentration of blueberry stem or leaf extract and the plaque reduction rate. In the figure, (a) shows the test results when blueberry stem extract was added, and (b) shows the test results when blueberry leaf extract was added.

[0064] As shown in Figure 2, blueberry stem extract F. 7 and leaf extract F. 7 inhibited plaque formation by SARS-CoV-2 in a concentration-dependent manner. 50 ) Fr. 7 of the blueberry stem extract was 4.78 μg / mL, and Fr. 7 of the leaf extract was 3.46 μg / mL. Furthermore, the viral infectivity titer reduction rate at the biological safety concentration of 50 μg / mL was 99.89% for Fr. 7 of the blueberry stem extract and over 99.96% for Fr. 7 of the leaf extract.

[0065] <Test III: Mechanism of antiviral effect of processed blueberries> [III-1: Protease inhibitory activity test] Coronaviruses (CoVs) cause multiple respiratory and gastrointestinal infections, primarily in humans and animals. 3CL protease, also known as main protease (Mpro), plays a key role in processing the polyprotein translated from viral RNA. 3CL protease inhibitors that can prevent viral replication may be promising candidates for drugs that can be used to treat patients suffering from viral infections caused by SARS-CoV-2. Therefore, in this study, we evaluated the 3CL protease inhibitory activity of processed blueberries using a 3CL protease assay kit.

[0066] 3CL protease assay buffer, recombinant 3CL protease (MBP-tag) solution, protease substrate solution, and GC376 (known protease inhibitor) solution were prepared according to the protocol of the 3CL protease assay kit (BPS Biosciences). Blueberry stem extract fractions 1 and 7, and blueberry leaf extract fractions 1 and 7, were dissolved in DMSO to prepare 100x concentrated test solutions. The 100x concentrated test solutions were diluted with 3CL protease assay buffer to prepare test solutions. 30 μL of recombinant 3CL protease (MBP-tag) solution was dispensed into each well of an assay microplate. 30 μL of 3CL protease assay buffer was dispensed into blank wells containing no enzyme. 10 μL of GC376 solution was dispensed into wells containing known protease inhibitor controls. 10 μL of sample solution was dispensed into the sample wells. 10 μL of 3CL protease assay buffer was dispensed into the positive control wells containing no inhibitor or sample. The microplate was pre-incubated at room temperature for 30 minutes with gentle shaking. Then, 10 μL of protease substrate solution was dispensed into each well. The microplate was sealed and incubated at room temperature overnight (16–20 hours). The fluorescence intensity of each well was then measured using a microplate reader (excitation wavelength: 360 nm, emission wavelength: 460 nm). The protease activity of the sample was calculated by defining the enzyme-free blank as 0% protease activity and the inhibitor- and sample-free positive control as 100% protease activity. Figure 3 shows the relationship between blueberry stem or leaf extract concentration and 3CL protease activity. In the figure, A shows the test results when Fr. 7 of blueberry stem extract and leaf extract was added, and B shows the test results when Fr. 1 of blueberry stem extract and leaf extract was added. In A and B, (a) shows the test results when blueberry stem extract was added, and (b) shows the test results when blueberry leaf extract was added. The horizontal axis shows the logarithm of the extract concentration (log (μg / mL)), and the vertical axis shows protease activity (%).

[0067] As shown in Figure 3A, blueberry stem extract F. 7 and leaf extract F. 7 inhibited 3CL protease activity in a concentration-dependent manner. The 50% inhibitory concentration (IC 50 ) Fr. 7 of the blueberry stem extract was 15.2 μg / mL, and Fr. 7 of the blueberry leaf extract was 14.4 μg / mL. In contrast, as shown in Figure 3B, Fr. 1 of the blueberry stem extract slightly inhibited 3CL protease activity, but Fr. 1 of the blueberry leaf extract did not. These results suggest that processed blueberries may suppress viral growth by inhibiting the activity of 3CL protease, which is involved in processing the polyprotein translated from viral RNA.

[0068] [III-2: Plaque formation inhibition test (3)] To investigate the timing of the onset of the antiviral effect of blueberry processing, the timing of adding the sample solution was changed in the plaque formation inhibition test procedure of Test II-1. The inoculated virus and host cells were the same as those in Test II-1. The host cells were inoculated at 1 x 10 in a 12-well plate containing MEM medium. 5 The cells were seeded at a cell density of 1 × 10 cells / mL and cultured for 24 hours. After that, the culture medium was removed and 1 × 10 cells were cultured. 4500 μL of the inoculum, prepared by diluting the PFU virus stock solution 10-fold or 100-fold, was added to each well. For untreated controls, the same volume of fresh medium was added instead of the inoculum. The test sample was added (i) 5 hours before virus infection (hereafter also referred to as the "Pre" treatment), (ii) 2 hours after virus infection (adsorption) (hereafter also referred to as the "On" treatment), (iii) 3 days after virus infection (hereafter also referred to as the "Post" treatment), or (iv) at the start of all periods (i) through (iii) (hereafter also referred to as the "All" treatment). The culture medium was removed from each well and 500 μL of fresh medium containing a 50 μg / mL DMSO solution of the test sample was added. At the end of periods (i) through (iv), the culture medium was removed from each well, the cell surface was washed with PBS, and fresh medium was added to continue the culture and virus infection. Thereafter, the number of plaques was measured using the same procedure as in Test II-1, and the virus titer (PFU / mL) was calculated. The percentage of the virus titer in the sample added relative to the virus titer in the control, to which the same amount of DMSO was added instead of the sample, was calculated as the plaque reduction rate (%). The relationship between the timing of blueberry stem or leaf extract addition and the virus titer is shown in Figure 4, and the relationship between the timing of blueberry stem or leaf extract addition and the plaque reduction rate is shown in Figure 5. In Figure 4, the horizontal axis represents the timing of blueberry stem or leaf extract addition, and the vertical axis represents the logarithm of the virus titer (log(PFU / mL)). In Figure 5, the horizontal axis represents the timing of blueberry stem or leaf extract addition, and the vertical axis represents the plaque reduction rate (%).

[0069] As shown in Figures 4 and 5, when blueberry stem or leaf extract was added in the pre-treatment or on-treatment, the virus titer was significantly reduced and a high plaque reduction rate was observed. In contrast, when blueberry stem or leaf extract was added in the post-treatment, the virus titer was not significantly reduced and a low plaque reduction rate was observed. These results suggest that processed blueberries can strongly inhibit virus entry into host cells.

[0070] The present invention is not limited to the above-described embodiments, but includes various modifications. For example, the above-described embodiments have been described in detail to clearly explain the present invention, and the present invention is not necessarily limited to those including all of the described configurations. Furthermore, it is possible to add, delete, and / or replace part of the configuration of each embodiment with other configurations.

Claims

1. An antiviral agent against the new coronavirus (SARS-CoV-2) containing processed blueberries as an active ingredient, The antiviral agent as described above, wherein the processed blueberry product is a fraction obtained by applying a hot water extract of crushed blueberry stems or leaves to Sephadex (registered trademark) LH-20 column chromatography and eluting with an eluent of distilled water:acetone=3:

2.

2. 2. The antiviral agent according to claim 1, for preventing or treating one or more viral infections selected from the group consisting of viral pneumonia, viral myocarditis, viral encephalitis, viral vasculitis, and viral conjunctivitis caused by the novel coronavirus (SARS-CoV-2).

3. A food product containing processed blueberries as an active ingredient for preventing or treating symptoms or disorders in subjects with a viral infection caused by the new coronavirus (SARS-CoV-2), The food product, wherein the processed blueberry product is a fraction obtained by applying a hot water extract of crushed blueberry stems or leaves to Sephadex (registered trademark) LH-20 column chromatography and eluting with an eluent of distilled water:acetone=3:2.

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