WO2007099915A1 - Antiviral agent - Google Patents

Antiviral agent Download PDF

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Publication number
WO2007099915A1
WO2007099915A1 PCT/JP2007/053538 JP2007053538W WO2007099915A1 WO 2007099915 A1 WO2007099915 A1 WO 2007099915A1 JP 2007053538 W JP2007053538 W JP 2007053538W WO 2007099915 A1 WO2007099915 A1 WO 2007099915A1
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WO
WIPO (PCT)
Prior art keywords
hop
antiviral agent
cold water
water extract
influenza virus
Prior art date
Application number
PCT/JP2007/053538
Other languages
French (fr)
Japanese (ja)
Inventor
Syuichi Segawa
Hisako Yasui
Original Assignee
Sapporo Breweries Limited
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Filing date
Publication date
Application filed by Sapporo Breweries Limited filed Critical Sapporo Breweries Limited
Priority to JP2008502775A priority Critical patent/JP5171614B2/en
Publication of WO2007099915A1 publication Critical patent/WO2007099915A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention relates to an antiviral agent.
  • EGCG green tea component epicarocatechin gallate
  • HTFDG tea component theaflavin galley HTFDG
  • Hops include antioxidant bituminous glycosides such as humulone and lubron (Patent Literatures 1 to 3), and flavonoid glycosides such as rutin and isoquercitrin. It has been reported that it can be used as an antioxidant for foods and beverages and cosmetics (Patent Document 4).
  • Patent Document 1 Japanese Patent Laid-Open No. 04-202138
  • Patent Document 2 Japanese Patent Laid-Open No. 06-025081
  • Patent Document 3 Japanese Patent Laid-Open No. 06-312924
  • Patent Document 4 Japanese Patent Application Laid-Open No. 09-2917
  • Non-Patent Document 1 Nakayama et al., 1996, Journal of Infectious Diseases, 70 ⁇ , No. 11, 1190-1192
  • Non-Patent Document 2 Nakayama et al., 1996, Journal of Infectious Diseases, 68 ⁇ , No. 7, p824-828 Disclosure of the invention
  • hop pellets used for brewing effervescent alcoholic beverages such as beer (referred to dried hop blossoms, sieved after pulverization, and solidified after passing through a sieve)
  • hop extract extract extracted from hop blossoms, extracted from organic solvent or supercritical fluid
  • brewing sparkling alcoholic beverages such as beer Tissue or hop residues
  • secondary use of unused hops or hop residues that are discarded is not profitable in terms of cost, and there is a demand for higher usage and usage!
  • an object of the present invention is to provide a highly safe antiviral agent derived from a plant. Another object of the present invention is to provide foods and drinks, feeds and feed additives containing the above-mentioned antiviral agents, and pharmaceuticals containing the above antiviral agents as active ingredients. Means for solving the problem
  • the present invention provides an antiviral agent characterized by comprising a cold water extract of hop tissue as an active ingredient.
  • the extract of hop tissue has a cold water extract strength, particularly effectively suppressing chicken hemagglutination caused by influenza virus and suppressing viral infection of animal cells.
  • Hops are mainly used for brewing beer and have long been used for various purposes other than brewing. Therefore, anti-viral agents derived from hops are considered to have excellent safety to the human body.
  • the antiviral agent of the present invention is obtained by extracting hop tissue with cold water, it is not affected by heat and can stably maintain the activity of the antiviral agent.
  • the hop structure is a pulverized product of dried hop camellia, and is also a pulverized product of dried hop camellia, which is at least part of a pulverized product having a size equal to or less than the size of lupulin. It is preferable that is removed.
  • the hops used in brewing effervescent alcoholic beverages such as beer are the power to dry coconuts excluding stems and leaves, sieve them after pulverization, solidify those that have passed through the sieves, and use them as hop pellets The crushed material that does not pass through the sieve is discarded.
  • This discarded pulverized product is obtained by removing at least a part of the pulverized product having a size smaller than that of Lubrin, and most of it is hop cake. Contributes to the reduction of industrial waste and makes effective use of hop dredging.
  • the hop tissue may be a hop residue obtained by removing at least a part of a substance extracted from an organic solvent or a supercritical fluid from a dried hop spikelet.
  • hop spikelets used in beer brewing are used for hop extract extraction, but the hop residue remaining after hop extract extraction is discarded.
  • This discarded hop residue is obtained by removing at least a part of the substance extracted by organic solvent extraction or supercritical fluid, so if this is used as a raw material for extraction of antiviral agents, industrial waste will be reduced. Can contribute.
  • the dried hop spikelet pulverized product is preferably a pulverized product of dried hop spikelet frozen product.
  • the efficiency of the pulverization increases and the effect of heat generated during the pulverization is reduced, so that the activity of the antiviral agent can be stably maintained.
  • the pulverized product having a size smaller than the size of rubulin easily passes through the sieve, the purity of the pulverized product exceeding the size of rubulin is increased, and the purity of the component that affects the antiviral activity is also increased. it can.
  • the antiviral agent of the present invention includes astragalin, astragalin malonyl darcoside, isocercitrin, isoquercitrin malo rudarcoside, quercetin malo rudarcoside, kaempferol rutinoside, kenferrol malo rudarcoside, rutin And at least one, preferably at least two, more preferably all flavonoid glycosides selected from the group consisting of fluorosilenone glycosides.
  • the antiviral agent of the present invention preferably contains 0.001% to 5% by weight of each of these flavonoid glycosides.
  • the fluorosilphenone glycoside includes fluoroisobutyral phenone dalcoside, fluoro-2-methylbutyrophenone dalcoside, and fluoroisovale phenone dalcoside.
  • Influenza virus growth begins with the binding of hemagglutinin (hemadaltun protein) to the surface receptors of host cells.
  • hemagglutinin hemadaltun protein
  • Glycoproteins or glycolipids with N-acetylneuraminic acid serve as receptors.
  • the antiviral agent of the present invention contains at least one of the above flavonoid glycosides, it can be expected to be adsorbed on the hemagglutinin protein of influenza virus, and for this reason, it can be adsorbed to the cell surface receptor of influenza virus. It is thought that the binding is inhibited and the entry of influenza virus into the cell is prevented.
  • the antiviral agent is preferably an anti-influenza virus agent.
  • the antiviral agent Since the antiviral agent has an activity of suppressing the influenza hemagglutination reaction of influenza virus and inhibiting the infection of animal cells, it prevents infection of influenza virus in humans and animals, especially poultry, particularly chickens. It is suitable for preventing.
  • the antiviral agent of the present invention described above is excellent in antiviral activity and safety, it can be used as an active ingredient by being contained in foods, drinks, feeds, feed additives or pharmaceuticals.
  • the invention's effect is excellent in antiviral activity and safety, it can be used as an active ingredient by being contained in foods, drinks, feeds, feed additives or pharmaceuticals. The invention's effect
  • an antiviral agent which has an activity of suppressing viral chicken platelet aggregation reaction and can prevent infection of humans and animals with pathogenic viruses. Since the antiviral agent of the present invention is derived from a natural plant, it is excellent in safety, and each person can prevent infection with pathogenic virus in the home and lead to early healing after infection.
  • the antiviral agent of the present invention is produced as a by-product in the brewing of effervescent alcoholic beverages such as beer, and can use hops discarded as raw materials, contributing to the reduction of industrial waste, Can be used effectively by increasing the added value.
  • FIG. 1 is a simplified diagram showing the procedure of a chicken erythrocyte aggregation titer measurement test.
  • FIG. 2 is a graph showing the inhibitory action of a hop pellet cold water extract and a bent hop cold water extract on the hemagglutination action of influenza virus.
  • FIG. 3 is a diagram schematically showing the procedure of an infection inhibition test for animal cells of influenza virus.
  • FIG. 4 is a graph showing the inhibitory action of hop pellet cold water extract and Svent hop cold water extract on infection of influenza virus to MDCK cells.
  • FIG. 6 is a graph showing the infection rate of influenza virus in mice of each group.
  • FIG. 7 shows the survival rate of mice in each group.
  • the hop used in the present invention may be any hop varieties, but beer brewing hop varieties such as Czech Zapp, German Haratau tradition and domestic Furano No. 18 are preferred. In order to obtain a hop extract with a high antiviral activity, it is more preferable than the Czech zazat.
  • the hop organization in the present invention means any hop organization or a part thereof.
  • the hop tissue used for cold water extraction is more preferably a hop cocoon that prefers a cocoon that uses any of leaves, stems, and spikelets.
  • the hop bud is a cocoon leaf constituting the cocoon flower, and can be obtained by removing at least a part of the rubulin portion (yellow granule) from the cocoon flower.
  • the hop tissue used for cold water extraction of the present invention may be a hop koji that is discarded without being pulverized to a specified size when processing hop pellets used for brewing foaming alcoholic beverages such as beer. It may be a hop residue remaining after the hop blossoms described later are extracted with a supercritical fluid or an organic solvent.
  • the antiviral agent of the present invention contains a cold water extract of hop tissue as an active ingredient, and this active ingredient is obtained by a production method comprising a step of extracting a hop tissue with cold water.
  • the hop tissue is extracted with cold water
  • “cold water” means water at room temperature or lower, and usually refers to water at temperatures above 0 ° C and below 30 ° C.
  • the temperature of chilled water is preferably 5 ⁇ 3 ° C (more preferably 5 ⁇ 2 ° C), more preferably over 0 ° C and below 10 ° C.
  • extraction efficiency can be increased by adding a small amount of alcohol, preferably ethanol, to 10% by weight or less to water.
  • Hop tissue strength As a method of extracting an antiviral agent, natural products are extracted from plants with water. For example, there may be mentioned a method in which a hop structure and a certain amount of cold water are put in a container, allowed to stand for a predetermined time with appropriate stirring, and the extract is filtered to remove the residue. In order to completely remove impurities and impurities contained in the mixture, the supernatant obtained by further centrifuging the filtered extract (hereinafter, centrifuged supernatant) can be used as an antiviral agent. In addition, the obtained antiviral agent can be concentrated and dried for use.
  • the antiviral agent which is a cold water extract of hop tissue
  • a synthetic adsorbent examples include Amberlit e XAD-4, 7 and 16 (organo), activated carbon, and polybulupolypyrrolidone (PVPP; polyphenol adsorbent).
  • Amberlite XAD-4 is preferably used.
  • the water extract of hop tissue is passed through a column packed with a synthetic adsorbent, and the adsorbed component is eluted with, for example, a mixed solvent of water and methanol, and the eluted fraction is used as an antiviral agent. Can be used.
  • the antiviral agent of the present invention comprises a cold water extract of dried hop koji crushed material as an active ingredient. It is preferable to use a cold water extract from which at least a part of the pulverized product is removed as an active ingredient.
  • the pulverized product of dried hop spikelets used for cold water extraction includes, for example, a drying step of drying hop spikelets to obtain dried hop spikelets, and a pulverization step of pulverizing dried hop spikelets to obtain a pulverized product. And a sorting step for removing pulverized material having a size equal to or smaller than the size of lupulin.
  • the hop spikelets are dried at a temperature of 100 ° C or lower and the water content can be removed to such an extent that the hop spikelets can be stored. It is preferred to dry to ⁇ 9%.
  • the hop spikelets can be efficiently pulverized into a fine powder.
  • a pulverizer such as a pin mill, a hammer mill, or a ball mill may be used.
  • the sorting step the pulverized product is sieved, and for example, a pulverized product having a major axis of 0.1 mm or more can be selected as “larger than Lubrin”.
  • the major axis not to pass through the sieve to a major axis of 0.3 mm or more, more preferably a major axis of 0.5 mm or more.
  • a major axis of 0.5 mm or more it is more preferable to set the major axis not to pass through the sieve to a major axis of 0.3 mm or more, more preferably a major axis of 0.5 mm or more.
  • the cold water extract of the dried hop camellia powder obtained by removing at least a part of the pulverized product having a size smaller than the size of Lubrin is extracted with the above-mentioned cold water. Extract it using the method described in the step.
  • the dried hop spikelet pulverized product used for preparing the antiviral agent of the present invention is preferably a dried hop spikelet frozen product pulverized product.
  • the method for freezing dried hop spikelets is not particularly limited, but is preferably 10 ° C or lower, more preferably 35 ° C or lower.
  • the antiviral agent of the present invention is a hop residue cold water extract obtained by removing at least part of a substance extracted from an organic solvent or a supercritical fluid from dried hop spikelets. It can be.
  • the organic solvent used for the organic solvent extraction include alcohol and hexane, and ethanol having lower alcohol having 1 to 4 carbon atoms is more preferable.
  • the supercritical fluid used for the supercritical fluid extraction include carbon dioxide, water, methane, ethane, ethylene, propane, pentane, methanol, and ethanol, with carbon dioxide and carbon being preferred.
  • the antiviral agent of the present invention has an effect of preventing infection of various pathogenic viruses, and is suitable for preventing infection of airborne viruses.
  • airborne viruses include rhinovirus, coronavirus, adenovirus, enterovirus, influenza virus, parainfluenza virus, SARS coronavirus, RS virus, echovirus, simple herpes virus, EB virus, Examples include measles virus and cytomegalovirus, which are more suitable for preventing influenza virus infection.
  • the antiviral agent of the present invention can be added to foods and drinks, feeds or feed additives for the purpose of preventing infection with pathogenic viruses and alleviating symptoms, or preventing infection with pathogenic viruses or It can be used as an active ingredient of a pharmaceutical agent as a therapeutic agent.
  • food and drink refers to food and drink consumed by humans
  • feed refers to food and drink consumed by animals other than humans.
  • feed additive refers to what is added to such “feed”.
  • the above-mentioned food and drink, feed, feed additive and medicine have a hop tissue cold water extract.
  • a cold-hop extract of hop tissue or a freeze-dried product of this cold-water extract can be used as it is as the food, feed, feed additive or pharmaceutical product.
  • the food / beverage product, feed, and feed additive of the present invention contain the antiviral agent described above, and may contain an additive that is usually used in the field.
  • the additives include apple fiber, soybean fiber, meat extract, black vinegar extract, gelatin, corn starch, honey, animal and vegetable oils and fats, monosaccharides such as glucose, disaccharides such as sucrose, fructose and mannitol, dextrose and Examples include polysaccharides such as starch, sugar alcohols such as erythritol, xylitol and sorbitol, and vitamins such as vitamin C. These additives may be used alone or in combination.
  • the pharmaceutical agent of the present invention contains the above-described antiviral agent as an active ingredient, and may further contain a pharmaceutically acceptable additive.
  • the additives include monosaccharides such as glucose, disaccharides such as sucrose, fructose and mannitol, polysaccharides such as dextrose and starch, sugar alcohols such as erythritol, xylitol and sorbitol, vitamin C and the like.
  • Vitamins may be a single species or a plurality of species.
  • rub pellets rich in rubulin produced from dried hop spikelets
  • Svent hop force which is a fraction that has been partially removed, prepared cold water extracts (hereinafter referred to as “hop pellet cold water extract” and “svent hop cold water extract”, respectively).
  • the hops are dried at 50 ° C until the water content becomes 8%, pulverized with a dedicated pulverizer, and the hop structure that has passed through a sieve with a mesh opening of 0.3 mm is made into hop pellets, and this sieve is passed through. None, 0.3 mm or more yarn and weave was used as a vent hop. That is, the hop pellet is a fraction containing fine powder of hop tissue and rubulin, and the hop cocoon is a pulverized product of 0.3 mm or more of the cocoon leaf part constituting the coconut flower, and the pudding is removed from the pudding. Minutes.
  • the hop pellets or the bent hops thus obtained were suspended in cold water so as to be 5%, and left in a low-temperature room at 4 ° C for 1 hour to extract cold water. Thereafter, each cold water extract was filtered (ADVANTEC No. 5A) to remove the residue and freeze-dried.
  • Hop pellet cold water extract and Svent hop cold water extract are quantified for polyphenol concentration according to the foreign thiocult method shown below, and based on this concentration, chicken hemagglutination assay described later and infection of animal cells with influenza virus An inhibition test was conducted.
  • each cold water extract was dissolved in a phosphate buffer solution (hereinafter, 0.1% BSA-PBS) containing 0.1% ushi serum albumin so as to have a concentration of 0.05 mgZmL.
  • the phenol reagent was removed and allowed to react for 3 minutes.
  • an equivalent amount of 10% aqueous sodium carbonate solution was added to the added 1N phenol, and the mixture was further reacted at room temperature for 1 hour, and the absorbance at 700 nm was measured.
  • a calibration curve was prepared using a standard solution of gallic acid whose concentration was previously divided, and the concentration of polyphenol in each cold water extract was calculated as the gallic acid equivalent from this calibration curve.
  • Example 1 Inhibition of erythrocyte aggregation by cold water extract of hop tissue:
  • Influenza virus has hemagglutinin (HA), and has the effect of agglutinating erythrocytes in birds. Therefore, the presence or absence of influenza virus in the sample can be determined by examining the presence or absence of chicken erythrocyte agglutination.
  • the infection and propagation of influenza virus in humans and animals begins with the binding of viral surface HA to receptors on the host cell surface. Host details Influenza virus adsorbed on the cell surface is taken up into the cell by endocytosis, grows in the cell using the translation system of the host cell, and repeatedly infects other cells. For this reason, it is thought that if the binding of influenza virus to animal cells via HA is suppressed, infection of humans and animals with influenza virus can be prevented. So, hop yarn and
  • the following chicken erythrocyte agglutination titration measurement test was conducted to determine whether or not the woven cold water extract had an inhibitory effect on chicken erythrocyte aggregation.
  • Hop pellet cold water extract and Svent hop cold water extract are each thread and weave cold water extraction as described above, 0.1% BS so that the polyphenol concentration becomes a predetermined concentration after lyophilization.
  • the erythrocyte suspension was prepared by suspending chicken stock blood in 0.1% BSA'PBS, washing the erythrocytes by repeating the centrifugation at 3000rpm for 5 minutes three times, and then adding the precipitated erythrocyte fraction to the sedimented erythrocyte fraction. for
  • a new 0.1% BSA'PBS was prepared so as to be 0.5% (vZv).
  • the AZPuerto RicoZ8Z34 (PR8: HlNl) strain was used as the influenza virus strain.
  • Influenza virus solution is diluted with 0.1% BSA'PBS so that the TCID value is 0 5 8
  • TCID value causes virus infection in 50% of cultured cells
  • Fig. 1 is a simplified diagram showing the procedure of the chicken hemagglutination titer measurement test.
  • First add 50 ⁇ L of influenza virus solution and 50 ⁇ L of hop pellet cold water extract, subvent hop cold water extract, or 0.1% BSA'PBS to each well of a 96-well microplate, The reaction was allowed to proceed for 1 hour at room temperature. Thereafter, 2-fold serial dilution series (2 1-fold dilution - 2 15-fold dilution) made for each group by micropipette, these 50 L of 0. 5% (VZV) erythrocyte suspension at room temperature was added respectively The sample was allowed to stand for 1 hour, and the presence or absence of an agglutination image was observed.
  • VZV 0. 5%
  • the chicken hemagglutination reaction was determined to be positive when an hemagglutination image was observed, and the maximum dilution ratio at which the hemagglutination reaction was positive was determined as the hemagglutination titer (HA titer). Red
  • the presence or absence of hemagglutination-inhibiting activity is determined by comparing the HA titer (control group) of the dilution series obtained by reacting the erythrocyte suspension with a mixture of influenza virus solution and 0.1% BSA'PBS and the HA titer of each test extract. It investigated by comparing.
  • the values in the graph are the mean standard error of each experiment performed three times, and the Mann-Whitney test was used to test the significant difference between the HA value of the control group and the HA value of the test group.
  • FIG. 2 is a graph showing the hemagglutination-inhibiting action of the hop pellet cold water extract and the bent hop cold water extract on the HA titer of influenza virus.
  • the vertical axis represents the HA value, and the concentration of each test extract is represented by the polyphenol content.
  • the hop pellet cold water extract and the scavenged hop cold water extract significantly suppressed the HA titer of influenza virus at a concentration of 0.6 mg / mL. From this result, it was found that the cold tissue extract of hop tissue contains a substance that suppresses the hemagglutination reaction of influenza virus, suggesting that it has antiviral activity.
  • MDCK cells Melat-Darby canine kidney cells
  • MDCK cells are suitable for various virus infection experiments and are generally used for evaluating the infection inhibitory action of influenza virus.
  • the cells were cultured in MEM medium containing serum (FBS) and maintained by subculture twice a week.
  • FBS serum
  • a cell suspension of MDCK cells prepared in 3 ⁇ 10 5 cells ZmL was added to a 96-well plate at 200 ⁇ L, and cultured for 3 or 4 days.
  • Fig. 3 is a simplified diagram showing the procedure for an infection inhibition test for animal cells of influenza virus.
  • a 25 L influenza virus dilution (the above influenza The virus solution was diluted 5 ⁇ 10 4 times) and each concentration of hop pellet cold water extract or spent hop cold water extract was mixed in a 96-well plate and allowed to react at room temperature for 1 hour (hereinafter referred to as “this”).
  • the reaction solution is called a virus reaction solution;).
  • the MDCK cell force MEM medium previously cultured for 3 days or 4 days in a 96-well plate is removed by aspiration, and 25 ⁇ L of the virus reaction solution is added to the medium, and it is added in a 37 ° C CO incubator. The reaction was further continued for 1 hour.
  • maintenance medium (MEM medium containing 0.2% albumin and 5 gZmL acetyltilpsin) is added to each tool and cultured for 3 days.
  • the culture supernatant has the ability to agglutinate chicken erythrocytes. I investigated whether or not.
  • FIG. 4 is a graph showing the inhibitory action of the hop pellet cold water extract and the bent hop cold water extract on the infection of MDCK cells with influenza virus.
  • the vertical axis represents chicken hemagglutination inhibition rate (%), and the concentration of each test extract is represented by the polyphenol content.
  • the hop pellet cold water extract and the bent hop cold water extract inhibit infection and proliferation of MDCK cells by reacting with influenza virus for 1 hour at room temperature.
  • the benthic hop cold water extract has a concentration of 50 / z gZmL Inhibits more than 80% of infections and is stronger than hop pellet cold water extract at the same concentration.
  • the TCID of the subvent hop cold water extract was calculated to be 28.6 ⁇ gZmL.
  • Fig. 5 shows the results of LC MS analysis of flavonoids contained in the bent-hop chilled water extract.
  • astragalin, isoquercitrin, quercetin malonyl darcoside, kaempferol rutinoside, kenferrol malo-aldarcoside and rutin and the fluoroacylphenone glycoside, fluoroisobutyrophenone darcoside, fluoro-2-methylbutyrate. It was found to contain oral phenone dalcoside and fluoroisovalerophenone darcoside.
  • HPLC analysis conditions are as follows.
  • Table 2 shows the results of comparing the content ratios of quercetin and kaempferol before and after hydrolysis of the subvent hop cold water extract (%; weight ratio of each flavonoid aglycone to the dry weight of the hop cold water extract). Is.
  • the flavonoid aglycone containing quercetin and kaempferol was not detected before hydrolysis. After hydrolysis, it contained 1.02% quercetin and 1.21% kaempferol. There was found. Since the flavonoid aglycone was not detected in the Sventhop cold water extract before hydrolysis, the flavonoids contained in the Sventhop cold water extract exist as glycosides, contributing to antiviral activity! / It was suggested to speak.
  • the hop pellet cold water extract administration group contains a 0.1% BSA'PBS solution (hop pellet cold water extract administration solution) containing a hop pellet cold water extract and influenza virus, 0.1% BSA'PBS solution (svent hop cold water extract administration solution) containing Sventhop cold water extract and influenza virus, and the control group containing only influenza virus 0.1% BSA'PBS solution ( A control influenza virus administration solution) was administered to the nostrils of each mouse.
  • Influenza virus solution prepared by diluting 10 times with 1% BSAZPBS was used for the experiment.
  • the hop pellet cold water extract and the scavent hop cold water extract were prepared as described above, and dissolved in 0.1% BSAZPBS so that the polyphenol concentration was 50 mgZmL.
  • the hop pellet cold water extract administration solution and the subvent hop cold water extract administration solution used for infection of mice are 1. 98 mL of the hop pellet cold water extract and the spent hop cold water extract, respectively, and the above influenza virus solution. Were added at 0.02 mL each and reacted at 37 ° C for 3 minutes. A control solution for influenza virus! The above influenza virus solution was added to 0.1% BSAZPBS containing no hop tissue cold water extract and reacted at 37 ° C for 3 minutes.
  • mice were performed according to the following procedure. First, Nembutal was administered intraperitoneally to anesthetize the mice, and a hop pellet cold water extract administration solution and a spout were placed in both nostrils of each group of mice. 10 ⁇ L each (20 ⁇ LZ mouse) of the administration solution of the top hop cold water extract or the control influenza virus was administered. Every day from the start of administration, the changes in body weight of mice were recorded and observed for irregularities in hair and decreased motility, and the presence or absence of influenza virus infection and the number of survivors were examined. The presence or absence of influenza virus infection was determined to be established when two or more items were met, based on changes in mouse body weight, irregularities in hair, and decreased mobility.
  • FIG. 6 shows the infection rate of influenza virus in mice in each group
  • FIG. 7 shows the survival rate of mice in each group. The lower the infection rate and the higher the survival rate, the more the influenza virus infection was suppressed.
  • the hop pellet cold water extract administration group no infection with influenza virus was observed over the 2 weeks of the study period, and the survival rate was 100%.
  • the survival rate of the hop pellet cold water extract group was significantly higher than that of the control group, and a statistically significant difference ( ⁇ ⁇ 0.01) was observed between the two groups.
  • mice were observed on the third day of administration, and all mice were infected with influenza virus on the eighth day of administration. It was confirmed that the death of all mice did not die as in the control group, and 3 mice were alive even after 2 weeks.
  • the group treated with the cold pellet extract showed a higher survival rate compared to the control group, and a statistically significant difference was observed between the two groups ( ⁇ 0. 05).
  • the hop pellet cold water extract and the bent hop cold water extract effectively inhibit influenza virus infection.
  • Industrial applicability ADVANTAGE OF THE INVENTION According to this invention, it has the activity which suppresses the chicken platelet aggregation reaction of a virus, and the antiviral agent which can prevent the infection to the human and animal of pathogenic virus is provided. Since the antiviral agent of the present invention is derived from a natural plant, it is excellent in safety, and since it is produced as a by-product when brewing foaming alcoholic beverages such as beer and discarded, hops are used as raw materials. It contributes to the reduction of industrial waste and can be used effectively with increased added value of hops.

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Abstract

It is intended to provide a highly safe antiviral agent originating in a plant, a food and a drink containing this antiviral agent and a medicine containing this antiviral agent as the active ingredient. Namely, an antiviral agent characterized by containing, as the active ingredient, a cold water-extract of a hop tissue; and a food, a drink, a feed, a feed additive or a medicine characterized by containing this antiviral agent.

Description

明 細 書  Specification
抗ウィルス剤  Antiviral agent
技術分野  Technical field
[0001] 本発明は、抗ウィルス剤に関する。  [0001] The present invention relates to an antiviral agent.
背景技術  Background art
[0002] インフルエンザウイルスを初めとする病原性ウィルスは、ヒト又は動物への感染を繰 り返しながら伝播し、人々の健康を害する。近年では、病原性ウィルスのヒト又は動物 への感染を予防し、重篤な病気となるのを防ぐために、植物由来の天然物の利用が 注目されている。例えば、緑茶成分のェピガロカテキンガレート (EGCG) (非特許文 献 1)や紅茶成分のテアフラビンジガレー HTFDG) (非特許文献 2)は、インフルェ ンザウィルスのプラーク形成を抑制し、動物実験にお 、てマウスへのウィルス感染を 阻害することが報告されて 、る。  [0002] Pathogenic viruses such as influenza viruses are transmitted through repeated infections of humans or animals, and are detrimental to human health. In recent years, the use of plant-derived natural products has attracted attention in order to prevent infection of humans or animals with pathogenic viruses and prevent serious diseases. For example, the green tea component epicarocatechin gallate (EGCG) (Non-patent document 1) and the tea component theaflavin galley HTFDG (non-patent document 2) suppresses the formation of influenza virus plaques and is used in animal experiments. It has been reported to inhibit viral infection in mice.
[0003] 一方、ホップは、ビール等発泡性アルコール飲料の醸造において酵母や麦芽と並 ぶ重要な原料であるが、民間療法においては、鎮静剤ゃ抗催淫剤として使用される 。ホップには、抗酸化作用を有するフムロンやルブロン等の結晶性苦味配糖体 (特許 文献 1〜3)、ルチンやイソケルシトリン等のフラボノイド配糖体が含まれており、最近 では、ホップ中のポリフエノールカ 飲食品及び化粧品用の抗酸化剤として利用でき ることが報告されて!ヽる (特許文献 4)。  [0003] On the other hand, hop is an important raw material as well as yeast and malt in brewing effervescent alcoholic beverages such as beer, but in folk remedies, sedatives are used as anti-hypnotic agents. Hops include antioxidant bituminous glycosides such as humulone and lubron (Patent Literatures 1 to 3), and flavonoid glycosides such as rutin and isoquercitrin. It has been reported that it can be used as an antioxidant for foods and beverages and cosmetics (Patent Document 4).
特許文献 1：特開平 04 - 202138号公報  Patent Document 1: Japanese Patent Laid-Open No. 04-202138
特許文献 2：特開平 06 -025081号公報  Patent Document 2: Japanese Patent Laid-Open No. 06-025081
特許文献 3：特開平 06 - 312924号公報  Patent Document 3: Japanese Patent Laid-Open No. 06-312924
特許文献 4：特開平 09— 2917号公報  Patent Document 4: Japanese Patent Application Laid-Open No. 09-2917
非特許文献 1 :中山ら、 1996年、感染症学雑誌、 70卷、 11号、 1190〜1192 非特許文献 2 :中山ら、 1996年、感染症学雑誌、 68卷、 7号、 p824〜828 発明の開示  Non-Patent Document 1: Nakayama et al., 1996, Journal of Infectious Diseases, 70 卷, No. 11, 1190-1192 Non-Patent Document 2: Nakayama et al., 1996, Journal of Infectious Diseases, 68 卷, No. 7, p824-828 Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0004] し力しながら、植物由来の天然物で抗ウィルス活性を有するとして知られているもの は、上述した緑茶及び紅茶等由来のカテキン類に限られ、より広い範囲の病原性ゥ ィルスに対抗するには、他の植物種力 異なる抗ウィルス剤を探索することが望まれ ている。 [0004] However, it is a plant-derived natural product known to have antiviral activity Is limited to the above-mentioned catechins derived from green tea and black tea, and in order to combat a wider range of pathogenic viruses, it is desired to search for other antiviral agents having different plant species.
[0005] また、ビール等発泡性アルコール飲料の醸造に使用されるホップペレット（ホップ毬 花を乾燥させ、粉砕後に篩分し、篩を通過して微粉となったものを固めたものをいう） 又はホップエキス (ホップ毬花から、有機溶媒抽出又は超臨界流体抽出された抽出 物を 、う）の製造過程あるいはビール等発泡性アルコール飲料の醸造過程で副産物 として大量に廃棄されるホップの未利用組織又はホップ残渣は、土壌改良用の肥料 や家畜の餌として二次利用されるが、これらの需要は廃棄される量に比べてはるかに 低ぐそのほとんどが産業廃棄物となるのが現状である。さらに、廃棄されるホップの 未利用組織又はホップ残渣の二次利用は、コストの面で採算が合わず、より付加価 値の高 、利用法が望まれて!/、る。  [0005] Further, hop pellets used for brewing effervescent alcoholic beverages such as beer (referred to dried hop blossoms, sieved after pulverization, and solidified after passing through a sieve) Or unused hops that are discarded in large quantities as by-products in the manufacturing process of hop extract (extract extracted from hop blossoms, extracted from organic solvent or supercritical fluid) or in the process of brewing sparkling alcoholic beverages such as beer Tissue or hop residues are secondarily used as fertilizers for soil improvement and livestock feed, but these demands are much lower than the amount discarded, and most of them are industrial waste. is there. In addition, secondary use of unused hops or hop residues that are discarded is not profitable in terms of cost, and there is a demand for higher usage and usage!
[0006] そこで、本発明の目的は、植物に由来する安全性の高い抗ウィルス剤を提供するこ とにある。本発明の目的はまた、上記抗ウィルス剤を含有する飲食品、飼料及び飼 料添加物並びに上記抗ウィルス剤を有効成分とする医薬品を提供することにある。 課題を解決するための手段  [0006] Therefore, an object of the present invention is to provide a highly safe antiviral agent derived from a plant. Another object of the present invention is to provide foods and drinks, feeds and feed additives containing the above-mentioned antiviral agents, and pharmaceuticals containing the above antiviral agents as active ingredients. Means for solving the problem
[0007] 上記目的を達成するため、本発明は、ホップ組織の冷水抽出物を有効成分とする ことを特徴とする抗ウィルス剤を提供する。  [0007] In order to achieve the above object, the present invention provides an antiviral agent characterized by comprising a cold water extract of hop tissue as an active ingredient.
[0008] 本発明者らは、ホップ組織の抽出物のうち冷水抽出物力 特に効果的にインフルェ ンザウィルスによって引き起こされる鶏赤血球凝集反応を抑制し、動物細胞へのウイ ルス感染を抑制することを見出した。ホップは、ビールの醸造に主に用いられ、醸造 以外の種々の用途にも古くから利用されている植物であるため、ホップ由来の抗ウイ ルス剤は人体に対する安全性が優れると考えられる。また、本発明の抗ウィルス剤は 、ホップ組織を冷水で抽出して得られるものであるため、熱による影響を受けず、抗ゥ ィルス剤の活性を安定的に維持できる。  [0008] The present inventors have found that the extract of hop tissue has a cold water extract strength, particularly effectively suppressing chicken hemagglutination caused by influenza virus and suppressing viral infection of animal cells. . Hops are mainly used for brewing beer and have long been used for various purposes other than brewing. Therefore, anti-viral agents derived from hops are considered to have excellent safety to the human body. In addition, since the antiviral agent of the present invention is obtained by extracting hop tissue with cold water, it is not affected by heat and can stably maintain the activity of the antiviral agent.
[0009] 上記ホップ組織は、乾燥されたホップ苞の粉砕物であることが好ましぐまた、乾燥 されたホップ毬花の粉砕物であって、ルプリンの大きさ以下の粉砕物の少なくとも一 部が除かれたものであることが好ましい。 [0010] ビール等発泡性アルコール飲料の醸造で使用するホップは、茎や葉を除いた毬花 を乾燥させ、粉砕後に篩分して篩を通過したものを固めてホップペレットとして使用す る力 篩を通過しなカゝつた粉砕物は廃棄されることになる。この廃棄される粉砕物は、 ルブリンの大きさ以下の粉砕物の少なくとも一部が除かれたものであり、その大部分 はホップ苞であるため、これを原料として抗ウィルス剤の抽出に用いれば産業廃棄物 の減少に貢献し、ホップ苞を有効利用できる。 [0009] Preferably, the hop structure is a pulverized product of dried hop camellia, and is also a pulverized product of dried hop camellia, which is at least part of a pulverized product having a size equal to or less than the size of lupulin. It is preferable that is removed. [0010] The hops used in brewing effervescent alcoholic beverages such as beer are the power to dry coconuts excluding stems and leaves, sieve them after pulverization, solidify those that have passed through the sieves, and use them as hop pellets The crushed material that does not pass through the sieve is discarded. This discarded pulverized product is obtained by removing at least a part of the pulverized product having a size smaller than that of Lubrin, and most of it is hop cake. Contributes to the reduction of industrial waste and makes effective use of hop dredging.
[0011] また、上記ホップ組織は、乾燥されたホップ毬花から、有機溶媒抽出又は超臨界流 体抽出される物質の少なくとも一部が除かれたホップ残渣とすることができる。ビール の醸造で使用するホップ毬花は、ホップペレットとして使用する他に、ホップエキスの 抽出のために使用されるが、ホップエキスを抽出した後に残るホップ残渣は廃棄され ることになる。この廃棄されるホップ残渣は、有機溶媒抽出又は超臨界流体で抽出さ れる物質の少なくとも一部が除かれたものであるため、これを原料として抗ウィルス剤 の抽出に用いれば産業廃棄物の減少に貢献できる。  [0011] Further, the hop tissue may be a hop residue obtained by removing at least a part of a substance extracted from an organic solvent or a supercritical fluid from a dried hop spikelet. In addition to being used as hop pellets, hop spikelets used in beer brewing are used for hop extract extraction, but the hop residue remaining after hop extract extraction is discarded. This discarded hop residue is obtained by removing at least a part of the substance extracted by organic solvent extraction or supercritical fluid, so if this is used as a raw material for extraction of antiviral agents, industrial waste will be reduced. Can contribute.
[0012] さらに、上記の乾燥されたホップ毬花の粉砕物は、乾燥されたホップ毬花の凍結物 の粉砕物であることが好ましい。乾燥されたホップ毬花を凍結後に粉砕すると、粉砕 効率が増し、粉砕時に生じる熱の影響を受けに《なるため、抗ウィルス剤の活性を 安定的に維持できる。また、ルブリンの大きさ以下の粉砕物が容易に篩を通過するよ うになるため、ルブリンの大きさを越える粉砕物の純度が高まり、抗ウィルス活性に影 響を与える成分の純度も高めることができる。  [0012] Furthermore, the dried hop spikelet pulverized product is preferably a pulverized product of dried hop spikelet frozen product. When the dried hop spikelets are pulverized after freezing, the efficiency of the pulverization increases and the effect of heat generated during the pulverization is reduced, so that the activity of the antiviral agent can be stably maintained. In addition, since the pulverized product having a size smaller than the size of rubulin easily passes through the sieve, the purity of the pulverized product exceeding the size of rubulin is increased, and the purity of the component that affects the antiviral activity is also increased. it can.
[0013] また、本発明の抗ウィルス剤は、ァストラガリン、ァストラガリンマロニルダルコシド、ィ ソケルシトリン、イソケルシトリンマロ-ルダルコシド、ケルセチンマロ-ルダルコシド、 ケンフェロールルチノシド、ケンフエロールマロ-ルダルコシド、ルチン及びフロロァシ ルフエノン配糖体力ゝらなる群より選ばれるフラボノイド配糖体の少なくとも一つ、好まし くは少なくとも二つ、より好ましくは総てを含有することが好ましい。また、本発明の抗 ウィルス剤は、これらのフラボノイド配糖体のそれぞれを、 0. 001重量％〜5重量％ 含有することが好ましい。なお、上記フロロァシルフエノン配糖体には、フロロイソブチ 口フエノンダルコシド、フロロ一 2—メチルブチロフエノンダルコシド及びフロロイソバレ 口フエノンダルコシドが含まれる。 [0014] インフルエンザウイルスの増殖は、赤血球凝集素（へマダルチュン蛋白質）が宿主 細胞の表面レセプターに結合することによって始まる力 A型及び B型インフルェン ザウィルスの場合には、糖鎖の非還元末端に N—ァセチルノイラミン酸を持つ糖蛋白 質又は糖脂質がレセプターとなる。本発明の抗ウィルス剤が、上記フラボノイド配糖 体の少なくとも一つを含有する場合は、インフルエンザウイルスのへマグルチニン蛋 白質に吸着することが期待でき、このためにインフルエンザウイルスの細胞表面レセ プターへの結合が阻害され、細胞内へのインフルエンザウイルスの侵入が阻止され ると考免られる。 [0013] Further, the antiviral agent of the present invention includes astragalin, astragalin malonyl darcoside, isocercitrin, isoquercitrin malo rudarcoside, quercetin malo rudarcoside, kaempferol rutinoside, kenferrol malo rudarcoside, rutin And at least one, preferably at least two, more preferably all flavonoid glycosides selected from the group consisting of fluorosilenone glycosides. The antiviral agent of the present invention preferably contains 0.001% to 5% by weight of each of these flavonoid glycosides. The fluorosilphenone glycoside includes fluoroisobutyral phenone dalcoside, fluoro-2-methylbutyrophenone dalcoside, and fluoroisovale phenone dalcoside. [0014] Influenza virus growth begins with the binding of hemagglutinin (hemadaltun protein) to the surface receptors of host cells. In the case of type A and type B influenza viruses, Glycoproteins or glycolipids with N-acetylneuraminic acid serve as receptors. When the antiviral agent of the present invention contains at least one of the above flavonoid glycosides, it can be expected to be adsorbed on the hemagglutinin protein of influenza virus, and for this reason, it can be adsorbed to the cell surface receptor of influenza virus. It is thought that the binding is inhibited and the entry of influenza virus into the cell is prevented.
[0015] 上記抗ウィルス剤は、抗インフルエンザウイルス剤であることが好ましい。  [0015] The antiviral agent is preferably an anti-influenza virus agent.
[0016] 上記抗ウィルス剤は、インフルエンザウイルスの鶏赤血球凝集反応を抑制し、動物 細胞への感染を阻害する活性を有するため、インフルエンザウイルスのヒト及び動物 、中でも家禽類、特に鶏への感染を防止するのに好適である。 [0016] Since the antiviral agent has an activity of suppressing the influenza hemagglutination reaction of influenza virus and inhibiting the infection of animal cells, it prevents infection of influenza virus in humans and animals, especially poultry, particularly chickens. It is suitable for preventing.
[0017] なお、上述した本発明の抗ウィルス剤は、抗ウィルス活性と安全性に優れることから 、飲食品、飼料、飼料添加物又は医薬品に含有させて有効成分として利用できる。 発明の効果 [0017] Since the antiviral agent of the present invention described above is excellent in antiviral activity and safety, it can be used as an active ingredient by being contained in foods, drinks, feeds, feed additives or pharmaceuticals. The invention's effect
[0018] 本発明によれば、ウィルスの鶏血小板凝集反応を抑制する活性を有し、病原性ウイ ルスのヒト及び動物への感染を防止できる抗ウィルス剤が提供される。本発明の抗ゥ ィルス剤は、天然の植物由来であるため、安全性に優れ、各人が家庭内で病原性ゥ ィルスの感染を予防し、感染後においては早期治癒に導くことができる。  [0018] According to the present invention, there is provided an antiviral agent which has an activity of suppressing viral chicken platelet aggregation reaction and can prevent infection of humans and animals with pathogenic viruses. Since the antiviral agent of the present invention is derived from a natural plant, it is excellent in safety, and each person can prevent infection with pathogenic virus in the home and lead to early healing after infection.
[0019] また本発明の抗ウィルス剤は、ビール等発泡性アルコール飲料の醸造の際の副産 物として産出され、廃棄されるホップを原料とできるため、産業廃棄物の減少に貢献 し、ホップの付加価値を高めて有効利用できる。  [0019] Further, the antiviral agent of the present invention is produced as a by-product in the brewing of effervescent alcoholic beverages such as beer, and can use hops discarded as raw materials, contributing to the reduction of industrial waste, Can be used effectively by increasing the added value.
図面の簡単な説明  Brief Description of Drawings
[0020] [図 1]鶏赤血球凝集価測定試験の手順を簡易的に示した図である。 [0020] FIG. 1 is a simplified diagram showing the procedure of a chicken erythrocyte aggregation titer measurement test.
[図 2]インフルエンザウイルスの赤血球凝集作用に及ぼすホップペレット冷水抽出物 及びスベントホップ冷水抽出物の抑制作用を示すグラフである。  FIG. 2 is a graph showing the inhibitory action of a hop pellet cold water extract and a bent hop cold water extract on the hemagglutination action of influenza virus.
[図 3]インフルエンザウイルスの動物細胞への感染阻害試験の手順を簡易的に示し た図である。 [図 4]インフルエンザウイルスの MDCK細胞への感染に及ぼすホップペレット冷水抽 出物及びスベントホップ冷水抽出物の阻害作用を示すグラフである。 FIG. 3 is a diagram schematically showing the procedure of an infection inhibition test for animal cells of influenza virus. FIG. 4 is a graph showing the inhibitory action of hop pellet cold water extract and Svent hop cold water extract on infection of influenza virus to MDCK cells.
[図 5]スベントホップ冷水抽出物に含まれるフラボノイドを LC MS分析した結果であ る。  [Fig. 5] LCMS analysis of flavonoids contained in the extract of cold bent hop water.
[図 6]各群のマウスへのインフルエンザウイルスの感染率を示した図である。  FIG. 6 is a graph showing the infection rate of influenza virus in mice of each group.
[図 7]各群のマウスの生存率を示した図である。  FIG. 7 shows the survival rate of mice in each group.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0021] 以下、本発明に係る抗ウィルス剤の好適な実施形態について説明する。 Hereinafter, preferred embodiments of the antiviral agent according to the present invention will be described.
[0022] 本発明に使用するホップは、いずれのホップ品種であってもよいが、チェコ産ザ一 ッ種、ドイツ産ハラタウ ·トラディション種及び国産フラノ 18号等のビール醸造用ホップ 品種が好ましぐ抗ウィルス活性の高いホップ抽出物を得るには、チェコ産ザーッ種 力 り好ましい。 [0022] The hop used in the present invention may be any hop varieties, but beer brewing hop varieties such as Czech Zapp, German Haratau Tradition and domestic Furano No. 18 are preferred. In order to obtain a hop extract with a high antiviral activity, it is more preferable than the Czech zazat.
[0023] 本発明におけるホップ組織とは、ホップの 、ずれかの組織又はその一部を意味す る。冷水抽出に用いるホップ組織は、葉、茎及び毬花のいずれでもよぐ毬花が好ま しぐホップ苞がより好ましい。ホップ苞は、毬花を構成する苞葉のことであって、毬花 からルブリン部分 (黄色の顆粒)の少なくとも一部を取り除いて得ることができる。この ため、本発明の冷水抽出に用いるホップ組織は、ビール等発泡性アルコール飲料の 醸造に用いるホップペレットを加工する際に規定の大きさに粉砕されずに廃棄される ホップ苞であってもよぐ後述するホップ毬花を超臨界流体又は有機溶媒で抽出した 後に残るホップ残渣であってもよ 、。  [0023] The hop organization in the present invention means any hop organization or a part thereof. The hop tissue used for cold water extraction is more preferably a hop cocoon that prefers a cocoon that uses any of leaves, stems, and spikelets. The hop bud is a cocoon leaf constituting the cocoon flower, and can be obtained by removing at least a part of the rubulin portion (yellow granule) from the cocoon flower. For this reason, the hop tissue used for cold water extraction of the present invention may be a hop koji that is discarded without being pulverized to a specified size when processing hop pellets used for brewing foaming alcoholic beverages such as beer. It may be a hop residue remaining after the hop blossoms described later are extracted with a supercritical fluid or an organic solvent.
[0024] 本発明の抗ウィルス剤は、ホップ組織の冷水抽出物を有効成分として含有するもの であり、この有効成分は、ホップ組織を冷水で抽出する工程を備える製造方法により 得られる。本工程では、ホップ組織を冷水で抽出するが、「冷水」とは室温以下の水 を意味し、通常、 0°C超 30°C以下の水のことをいう。冷水の温度は、 0°C超 10°C以下 が好ましぐ 5± 3°C (さらには 5± 2°C)がより好ましい。なお、抽出時間を短縮するた めには、水に少量のアルコール、好ましくはエタノールを、 10重量％以下添加して抽 出効率を上げることができる。  [0024] The antiviral agent of the present invention contains a cold water extract of hop tissue as an active ingredient, and this active ingredient is obtained by a production method comprising a step of extracting a hop tissue with cold water. In this process, the hop tissue is extracted with cold water, and “cold water” means water at room temperature or lower, and usually refers to water at temperatures above 0 ° C and below 30 ° C. The temperature of chilled water is preferably 5 ± 3 ° C (more preferably 5 ± 2 ° C), more preferably over 0 ° C and below 10 ° C. In order to shorten the extraction time, extraction efficiency can be increased by adding a small amount of alcohol, preferably ethanol, to 10% by weight or less to water.
[0025] ホップ組織力 抗ウィルス剤を抽出する方法としては、植物から天然物を水抽出す る方法が広く採用でき、例えば、ホップ組織と一定量の冷水とを容器に入れ、適宜撹 拌しながら所定時間静置し、抽出液を濾過して残渣を取り除く方法が挙げられる。混 入する残渣ゃ不純物等を完全に取り除くためには、濾過した抽出液をさらに遠心分 離すればよぐその上清 (以下、遠心上清)を抗ウィルス剤として使用できる。なお、 得られた抗ウィルス剤は、濃縮し、乾燥して使用することもできる。 [0025] Hop tissue strength As a method of extracting an antiviral agent, natural products are extracted from plants with water. For example, there may be mentioned a method in which a hop structure and a certain amount of cold water are put in a container, allowed to stand for a predetermined time with appropriate stirring, and the extract is filtered to remove the residue. In order to completely remove impurities and impurities contained in the mixture, the supernatant obtained by further centrifuging the filtered extract (hereinafter, centrifuged supernatant) can be used as an antiviral agent. In addition, the obtained antiviral agent can be concentrated and dried for use.
[0026] また、ホップ組織の冷水抽出物である抗ウィルス剤は、合成吸着剤を充填したカラ ムに通して、精製して使用することもできる。合成吸着剤としては、例えば、 Amberlit e XAD— 4、 7及び 16 (オルガノ社）、活性炭、ポリビュルポリピロリドン（PVPP ;ポリ フエノール吸着剤）が挙げられ、この中でも Amberlite XAD— 4が好ましく用いられ る。具体的には、ホップ組織の水抽出物を、合成吸着剤を充填したカラムに通し、そ の吸着成分を、例えば、水及びメタノールの混合溶媒で溶出させ、溶出した画分を 抗ウィルス剤として使用できる。  [0026] The antiviral agent, which is a cold water extract of hop tissue, can also be used after purification through a column filled with a synthetic adsorbent. Examples of the synthetic adsorbent include Amberlit e XAD-4, 7 and 16 (organo), activated carbon, and polybulupolypyrrolidone (PVPP; polyphenol adsorbent). Among them, Amberlite XAD-4 is preferably used. The Specifically, the water extract of hop tissue is passed through a column packed with a synthetic adsorbent, and the adsorbed component is eluted with, for example, a mixed solvent of water and methanol, and the eluted fraction is used as an antiviral agent. Can be used.
[0027] 本発明の抗ウィルス剤は、乾燥されたホップ苞の粉砕物の冷水抽出物を有効成分 とすることが好ましぐまた、乾燥されたホップ毬花の粉砕物からルブリンの大きさ以下 の粉砕物の少なくとも一部が除かれたものの冷水抽出物を有効成分とすることが好ま しい。冷水抽出に用いる乾燥ホップ毬花の粉砕物は、例えば、ホップ毬花を乾燥して 乾燥ホップ毬花を得る乾燥工程と、乾燥ホップ毬花を粉砕して粉砕物を得る粉砕ェ 程と、この粉砕物力ゝらルプリンの大きさ以下の粉砕物を取り除く選別工程と、を備える 製造方法により得られる。  [0027] It is preferable that the antiviral agent of the present invention comprises a cold water extract of dried hop koji crushed material as an active ingredient. It is preferable to use a cold water extract from which at least a part of the pulverized product is removed as an active ingredient. The pulverized product of dried hop spikelets used for cold water extraction includes, for example, a drying step of drying hop spikelets to obtain dried hop spikelets, and a pulverization step of pulverizing dried hop spikelets to obtain a pulverized product. And a sorting step for removing pulverized material having a size equal to or smaller than the size of lupulin.
[0028] 乾燥工程では、ホップ毬花を 100°C以下の温度で乾燥させ、ホップ毬花を保存可 能な程度にまで水分を除去できればよいが、 55°C以下の温度で水分含量を 7〜9% まで乾燥することが好ましい。粉砕工程では、ホップ毬花を効率的に微粉状に粉砕 できればよぐ例えば、ピンミル、ハンマーミル、ボールミル等の粉砕機を用いればよ い。選別工程では、粉砕物をふるいにかけ、例えば、長径が 0. 1mm以上の粉砕物 を「ルブリンを超える大きさ」のものとして選別することができる。この場合において、篩 を通過させない大きさを長径 0. 3mm以上とすることが好ましぐ長径 0. 5mm以上と することがより好ま 、。乾燥ホップ毬花の粉砕物カもルプリンの大きさ以下の粉砕物 を取り除くには、例えば、目開き 0. 1、0. 3又は 0. 5mmのふるいで乾燥ホップ毬花 の粉碎物をふるい [0028] In the drying process, it is sufficient that the hop spikelets are dried at a temperature of 100 ° C or lower and the water content can be removed to such an extent that the hop spikelets can be stored. It is preferred to dry to ~ 9%. In the pulverization step, it is sufficient if the hop spikelets can be efficiently pulverized into a fine powder. For example, a pulverizer such as a pin mill, a hammer mill, or a ball mill may be used. In the sorting step, the pulverized product is sieved, and for example, a pulverized product having a major axis of 0.1 mm or more can be selected as “larger than Lubrin”. In this case, it is more preferable to set the major axis not to pass through the sieve to a major axis of 0.3 mm or more, more preferably a major axis of 0.5 mm or more. To remove pulverized material that is smaller than the size of lupulin, for example, dry hop buds with 0.1, 0.3, or 0.5 mm sieves. Sifting powdered rice cake
分け、ふるいを通過しな力つた粉砕物を回収すればよい。なお、乾燥ホップ毬花の粉 砕物からルブリンの大きさ以下の粉砕物の少なくとも一部が除かれたものの冷水抽出 物は、こうして選別された乾燥ホップ毬花の粉砕物を、上述した冷水で抽出する工程 で記載した方法で抽出すればょ ヽ。  What is necessary is just to collect | recover the pulverized material which divided and passed through the sieve. In addition, the cold water extract of the dried hop camellia powder obtained by removing at least a part of the pulverized product having a size smaller than the size of Lubrin is extracted with the above-mentioned cold water. Extract it using the method described in the step.
[0029] さらに、本発明の抗ウィルス剤を調製するために使用する乾燥されたホップ毬花の 粉砕物は、乾燥されたホップ毬花の凍結物の粉砕物であることが好ましい。乾燥され たホップ毬花を凍結する方法は、特に制限されないが、 10°C以下が好ましぐ 3 5°C以下がより好ましい。  [0029] Further, the dried hop spikelet pulverized product used for preparing the antiviral agent of the present invention is preferably a dried hop spikelet frozen product pulverized product. The method for freezing dried hop spikelets is not particularly limited, but is preferably 10 ° C or lower, more preferably 35 ° C or lower.
[0030] また、本発明の抗ウィルス剤は、乾燥されたホップ毬花から、有機溶媒抽出又は超 臨界流体抽出される物質の少なくとも一部が除かれたホップ残渣の冷水抽出物を有 効成分とすることができる。有機溶媒抽出に用いる有機溶媒としては、例えば、アル コール又はへキサンが挙げられ、炭素数 1〜4の低級アルコールが好ましぐエタノー ルがより好ましい。超臨界流体抽出に用いる超臨界流体としては、例えば、二酸化炭 素、水、メタン、ェタン、エチレン、プロパン、ペンタン、メタノール、エタノールが挙げ られ、二酸ィ匕炭素が好ましい。  [0030] Further, the antiviral agent of the present invention is a hop residue cold water extract obtained by removing at least part of a substance extracted from an organic solvent or a supercritical fluid from dried hop spikelets. It can be. Examples of the organic solvent used for the organic solvent extraction include alcohol and hexane, and ethanol having lower alcohol having 1 to 4 carbon atoms is more preferable. Examples of the supercritical fluid used for the supercritical fluid extraction include carbon dioxide, water, methane, ethane, ethylene, propane, pentane, methanol, and ethanol, with carbon dioxide and carbon being preferred.
[0031] 本発明の抗ウィルス剤は、種々の病原性ウィルスの感染を防止する効果があり、空 気感染性ウィルスの感染を防止するのに適して 、る。空気感染性ウィルスとしては、 例えば、ライノウィルス、コロナウィルス、アデノウイルス、ェンテロウィルス、インフル ェンザウィルス、パラインフルエンザウイルス、 SARSコロナウィルス、 RSウィルス、ェ コーウィルス、単純へルぺスウィルス、 EBウィルス、麻疹ウィルス、サイトメガロウィル スが挙げられ、インフルエンザウイルスの感染を防止するのにより適している。  [0031] The antiviral agent of the present invention has an effect of preventing infection of various pathogenic viruses, and is suitable for preventing infection of airborne viruses. Examples of airborne viruses include rhinovirus, coronavirus, adenovirus, enterovirus, influenza virus, parainfluenza virus, SARS coronavirus, RS virus, echovirus, simple herpes virus, EB virus, Examples include measles virus and cytomegalovirus, which are more suitable for preventing influenza virus infection.
[0032] 本発明の抗ウィルス剤は、病原性ウィルスの感染の予防や症状の緩和を目的とし て飲食品、飼料又は飼料添加物に添加することができ、又は病原性ウィルスの感染 予防剤若しくは治療剤となる医薬品の有効成分として使用できる。ここで、「飲食品」 とは、ヒトが摂取する飲食品のことをいい、「飼料」とは、ヒト以外の動物が摂取する飲 食品のことをいう。また、「飼料添加物」とは、このような「飼料」に添加するもののことを いう。上記飲食品、飼料、飼料添加物及び医薬品は、ホップ組織の冷水抽出物を有 効成分とする抗ウィルス剤のみ力 なっていてもよぐ例えば、ホップ組織の冷水抽 出物又はこの冷水抽出物の凍結乾燥品をそのまま上記飲食品、飼料、飼料添加物 又は医薬品として使用できる。 [0032] The antiviral agent of the present invention can be added to foods and drinks, feeds or feed additives for the purpose of preventing infection with pathogenic viruses and alleviating symptoms, or preventing infection with pathogenic viruses or It can be used as an active ingredient of a pharmaceutical agent as a therapeutic agent. Here, “food and drink” refers to food and drink consumed by humans, and “feed” refers to food and drink consumed by animals other than humans. “Feed additive” refers to what is added to such “feed”. The above-mentioned food and drink, feed, feed additive and medicine have a hop tissue cold water extract. For example, a cold-hop extract of hop tissue or a freeze-dried product of this cold-water extract can be used as it is as the food, feed, feed additive or pharmaceutical product.
[0033] 本発明の飲食品、飼料、飼料添加物は、上記の抗ウィルス剤を含んでおり、当該分 野で通常使用される添加物を含んでいてもよい。この添加物としては、例えば、リンゴ ファイバー、大豆ファイバー、肉エキス、黒酢エキス、ゼラチン、コーンスターチ、蜂蜜 、動植物油脂、グルコース等の単糖類、スクロース、フルクトース及びマン-トール等 の二糖類、デキストロース及びデンプン等の多糖類、エリスリトール、キシリトール及 びソルビトール等の糖アルコール類、ビタミン C等のビタミン類が挙げられ、これらの 添加物は単独種又は複数種であってもよ 、。  [0033] The food / beverage product, feed, and feed additive of the present invention contain the antiviral agent described above, and may contain an additive that is usually used in the field. Examples of the additives include apple fiber, soybean fiber, meat extract, black vinegar extract, gelatin, corn starch, honey, animal and vegetable oils and fats, monosaccharides such as glucose, disaccharides such as sucrose, fructose and mannitol, dextrose and Examples include polysaccharides such as starch, sugar alcohols such as erythritol, xylitol and sorbitol, and vitamins such as vitamin C. These additives may be used alone or in combination.
[0034] 本発明の医薬品は、上記の抗ウィルス剤を有効成分として含んでおり、薬学的に許 容される添加物をさらに含んでいてもよい。この添加物としては、例えば、グルコース 等の単糖類、スクロース、フルクトース及びマン-トール等の二糖類、デキストロース 及びデンプン等の多糖類、エリスリトール、キシリトール及びソルビトール等の糖アル コール類、ビタミン C等のビタミン類、アカシアゴム、リン酸カルシウム、アルギン酸塩、 珪酸カルシウム、微結晶性セルロース、ポリビュルピロリドン、セルロース誘導体、トラ ガカント、ゼラチン、シロップ、ヒドロキシ安息香酸メチル、タルク、ステアリン酸マグネ シゥム、水、鉱油が挙げられ、これらの添加物は単独種又は複数種であってもよい。  [0034] The pharmaceutical agent of the present invention contains the above-described antiviral agent as an active ingredient, and may further contain a pharmaceutically acceptable additive. Examples of the additives include monosaccharides such as glucose, disaccharides such as sucrose, fructose and mannitol, polysaccharides such as dextrose and starch, sugar alcohols such as erythritol, xylitol and sorbitol, vitamin C and the like. Vitamins, gum acacia, calcium phosphate, alginate, calcium silicate, microcrystalline cellulose, polybutylpyrrolidone, cellulose derivatives, tragacanth, gelatin, syrup, methyl hydroxybenzoate, talc, magnesium stearate, water, mineral oil These additives may be a single species or a plurality of species.
[0035] さらに、病原性ウィルスの感染の予防や症状の緩和の目的で、食品添加物として、 特定保健用食品、特殊栄養食品、栄養補助食品、健康食品、機能性食品や病者用 食品等の飲食物に配合することもできる。  [0035] Furthermore, as food additives for the purpose of preventing infection with pathogenic viruses and alleviating symptoms, foods for specified health use, special nutritional foods, dietary supplements, health foods, functional foods, foods for the sick, etc. It can also be blended in food and drink.
実施例  Example
[0036] 以下、実施例を挙げて本発明を具体的に説明するが、本発明はこれらの実施例に 限定されるものではない。また、特に明記しない限り、「％」は「重量％」を表す。  Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to these examples. Unless otherwise specified, “%” represents “% by weight”.
[0037] (ホップ組織の冷水抽出物の調製）  [0037] (Preparation of cold water extract of hop tissue)
ホップ組織の冷水抽出物の抗ウィルス活性を調べるため、乾燥されたホップ毬花か ら製造された、ルブリンを豊富に含有するホップペレット（チェコ産ザーッ種: SSAタイ プタイプ 90)と、ホップペレットの製造過程で副産物として生じ、ルプリンの少なくとも 一部が取り除かれた画分であるスベントホップ力 それぞれ冷水抽出物（以下、それ ぞれ「ホップペレット冷水抽出物」及び「スベントホップ冷水抽出物」という）を調製したTo examine the antiviral activity of cold water extracts of hop tissue, rub pellets rich in rubulin (Czech zazat species: SSA type 90) produced from dried hop spikelets, As a by-product in the manufacturing process, Svent hop force, which is a fraction that has been partially removed, prepared cold water extracts (hereinafter referred to as “hop pellet cold water extract” and “svent hop cold water extract”, respectively).
。まず、ホップを 50°Cで水分含量が 8%になるまで乾燥させ、専用の粉砕機で粉砕し 、 目開き 0. 3mmのふるいを通過したホップ組織をホップペレットとし、このふるいを通 過しなかった 0. 3mm以上の糸且織をスベントホップとした。すなわち、ホップペレットは 、ホップ組織の微粉とルブリンを含有する画分であり、ホップ苞は、毬花を構成 する苞葉部分の 0. 3mm以上の粉砕物であって、ルプリンが取り除かれた画分であ る。 . First, the hops are dried at 50 ° C until the water content becomes 8%, pulverized with a dedicated pulverizer, and the hop structure that has passed through a sieve with a mesh opening of 0.3 mm is made into hop pellets, and this sieve is passed through. None, 0.3 mm or more yarn and weave was used as a vent hop. That is, the hop pellet is a fraction containing fine powder of hop tissue and rubulin, and the hop cocoon is a pulverized product of 0.3 mm or more of the cocoon leaf part constituting the coconut flower, and the pudding is removed from the pudding. Minutes.
[0038] こうして得られたホップペレット又はスベントホップは、 5%となるように冷水で懸濁し 、 4°Cの低温室で 1晚静置することにより冷水抽出した。その後、各冷水抽出物を濾 過 (ADVANTEC No. 5A)することにより残渣を取り除き、凍結乾燥して用いた。  [0038] The hop pellets or the bent hops thus obtained were suspended in cold water so as to be 5%, and left in a low-temperature room at 4 ° C for 1 hour to extract cold water. Thereafter, each cold water extract was filtered (ADVANTEC No. 5A) to remove the residue and freeze-dried.
[0039] (ポリフエノール濃度の定量）  [0039] (Quantification of polyphenol concentration)
ホップペレット冷水抽出物及びスベントホップ冷水抽出物は、以下に示すフォーリン チオカルト法に従ってポリフエノール濃度を定量し、この濃度を基準にして、後述する 鶏赤血球凝集価測定試験及びインフルエンザウイルスの動物細胞への感染阻害試 験を行った。  Hop pellet cold water extract and Svent hop cold water extract are quantified for polyphenol concentration according to the foreign thiocult method shown below, and based on this concentration, chicken hemagglutination assay described later and infection of animal cells with influenza virus An inhibition test was conducted.
[0040] まず、各冷水抽出物を 0. 05mgZmLとなるように 0. 1%ゥシ血清アルブミン含有リ ン酸緩衝液（以下、 0. 1%BSA-PBS)に溶解し、等量の 1Nフエノール試薬をカロえ、 3分間反応させた。その後、添加した 1Nフエノールと等量の 10%炭酸ナトリウム水溶 液を加えて、室温でさらに 1時間反応させ、 700nmの吸光度を測定した。その際、濃 度が予め分力ゝつて ヽる没食子酸の標準溶液を用いて検量線を作製し、この検量線か ら各冷水抽出物中のポリフ ノール濃度を没食子酸当量として算出した。  [0040] First, each cold water extract was dissolved in a phosphate buffer solution (hereinafter, 0.1% BSA-PBS) containing 0.1% ushi serum albumin so as to have a concentration of 0.05 mgZmL. The phenol reagent was removed and allowed to react for 3 minutes. Thereafter, an equivalent amount of 10% aqueous sodium carbonate solution was added to the added 1N phenol, and the mixture was further reacted at room temperature for 1 hour, and the absorbance at 700 nm was measured. At that time, a calibration curve was prepared using a standard solution of gallic acid whose concentration was previously divided, and the concentration of polyphenol in each cold water extract was calculated as the gallic acid equivalent from this calibration curve.
[0041] (実施例 1)ホップ組織の冷水抽出物の赤血球凝集抑制作用：  [0041] (Example 1) Inhibition of erythrocyte aggregation by cold water extract of hop tissue:
インフルエンザウイルスは、赤血球凝集素（hemagglutinin;以下、 HA)を持って おり、この HAにより-ヮトリの赤血球を凝集させる作用を有している。このため、鶏赤 血球凝集反応の有無を調べることにより、検体中のインフルエンザウイルスの有無を 判定できる。一方、インフルエンザウイルスのヒト及び動物への感染及び増殖は、ウイ ルス表面の HAが宿主細胞表面のレセプターに結合することによって始まる。宿主細 胞表面に吸着したインフルエンザウイルスはエンドサイト一シスにより細胞内に取り込 まれ、細胞内で宿主細胞の翻訳系を利用して増殖し、他の細胞への感染を繰り返す 。このため、 HAを介したインフルエンザウイルスの動物細胞への結合を抑制すれば 、インフルエンザウイルスのヒト及び動物への感染を防止できると考えられている。そ こで、ホップ糸且 Influenza virus has hemagglutinin (HA), and has the effect of agglutinating erythrocytes in birds. Therefore, the presence or absence of influenza virus in the sample can be determined by examining the presence or absence of chicken erythrocyte agglutination. On the other hand, the infection and propagation of influenza virus in humans and animals begins with the binding of viral surface HA to receptors on the host cell surface. Host details Influenza virus adsorbed on the cell surface is taken up into the cell by endocytosis, grows in the cell using the translation system of the host cell, and repeatedly infects other cells. For this reason, it is thought that if the binding of influenza virus to animal cells via HA is suppressed, infection of humans and animals with influenza virus can be prevented. So, hop yarn and
織の冷水抽出物に鶏赤血球凝集抑制作用があるかどうかについて、以下の鶏赤血 球凝集価測定試験を行って調べた。  The following chicken erythrocyte agglutination titration measurement test was conducted to determine whether or not the woven cold water extract had an inhibitory effect on chicken erythrocyte aggregation.
[0042] 1)実験材料 [0042] 1) Experimental materials
ホップペレット冷水抽出物及びスベントホップ冷水抽出物は、上述したように各糸且織 力 冷水抽出を行い、凍結乾燥後、所定のポリフエノール濃度となるように 0. 1%BS Hop pellet cold water extract and Svent hop cold water extract are each thread and weave cold water extraction as described above, 0.1% BS so that the polyphenol concentration becomes a predetermined concentration after lyophilization.
A · PBSで溶解して調製した。 A · Prepared by dissolving in PBS.
[0043] 赤血球浮遊液は、鶏保存血を 0. 1%BSA'PBSに縣濁し、 3000rpm、 5分間の遠 心分離操作を 3回繰り返して赤血球を洗浄し、その後、沈殿した赤血球画分に対して[0043] The erythrocyte suspension was prepared by suspending chicken stock blood in 0.1% BSA'PBS, washing the erythrocytes by repeating the centrifugation at 3000rpm for 5 minutes three times, and then adding the precipitated erythrocyte fraction to the sedimented erythrocyte fraction. for
0. 5% (vZv)になるように新しい 0. 1%BSA'PBSをカ卩えて調製した。 A new 0.1% BSA'PBS was prepared so as to be 0.5% (vZv).
[0044] インフルエンザウイルス株は、 AZPuerto RicoZ8Z34 (PR8 :HlNl)株を用い[0044] The AZPuerto RicoZ8Z34 (PR8: HlNl) strain was used as the influenza virus strain.
、インフルエンザウイルス液は、 TCID 値力 05· 8となるように 0. 1%BSA'PBSで希 Influenza virus solution is diluted with 0.1% BSA'PBS so that the TCID value is 0 5 8
50  50
釈して調製した。ここで、 TCID 値とは培養細胞の 50%にウィルス感染を起こさせる  Prepared. Here, TCID value causes virus infection in 50% of cultured cells
50  50
ウィルス希釈倍率のことである。  Virus dilution rate.
[0045] 2)鶏赤血球凝集価測定試験 [0045] 2) Chicken erythrocyte agglutination test
図 1は、鶏赤血球凝集価測定試験の手順を簡易的に示した図である。まず、 96穴 マイクロプレートの各ゥエルに 50 μ Lのインフルエンザウイルス液と 50 μ Lの各濃度 のホップペレット冷水抽出物、スベントホップ冷水抽出物又は 0. 1%BSA'PBSとを 加えて混合し、室温で 1時間反応させた。その後、マイクロピペットにより 2倍段階希 釈系列（21倍希釈〜 215倍希釈）をそれぞれの群について作り、これらに 50 Lの 0. 5% (vZv)赤血球浮遊液をそれぞれ加えて室温で 1晚静置し、赤血球凝集像の有 無を観察した。 Fig. 1 is a simplified diagram showing the procedure of the chicken hemagglutination titer measurement test. First, add 50 μL of influenza virus solution and 50 μL of hop pellet cold water extract, subvent hop cold water extract, or 0.1% BSA'PBS to each well of a 96-well microplate, The reaction was allowed to proceed for 1 hour at room temperature. Thereafter, 2-fold serial dilution series (2 1-fold dilution - 2 15-fold dilution) made for each group by micropipette, these 50 L of 0. 5% (VZV) erythrocyte suspension at room temperature was added respectively The sample was allowed to stand for 1 hour, and the presence or absence of an agglutination image was observed.
[0046] 鶏赤血球凝集反応は、赤血球凝集像が認められた場合に陽性であると判定し、赤 血球凝集反応が陽性となる最高希釈倍率を赤血球凝集価 (HA価)として求めた。赤 血球凝集抑制作用の有無は、インフルエンザウイルス液と 0. 1%BSA'PBSの混合 液に赤血球浮遊液を反応させた希釈系列の HA価 (対照群）と各被検抽出物の HA 価とを比較することにより調べた。グラフ中の値は、それぞれの実験を 3回行った平均 値士標準誤差で、対照群の HA価と試験群の HA価との間の有意差検定は、 Mann —Whitney検定で行った。 [0046] The chicken hemagglutination reaction was determined to be positive when an hemagglutination image was observed, and the maximum dilution ratio at which the hemagglutination reaction was positive was determined as the hemagglutination titer (HA titer). Red The presence or absence of hemagglutination-inhibiting activity is determined by comparing the HA titer (control group) of the dilution series obtained by reacting the erythrocyte suspension with a mixture of influenza virus solution and 0.1% BSA'PBS and the HA titer of each test extract. It investigated by comparing. The values in the graph are the mean standard error of each experiment performed three times, and the Mann-Whitney test was used to test the significant difference between the HA value of the control group and the HA value of the test group.
[0047] 図 2は、インフルエンザウイルスの HA価に及ぼすホップペレット冷水抽出物及びス ベントホップ冷水抽出物の赤血球凝集抑制作用を示すグラフである。縦軸は HA価 を表し、各被検抽出物の濃度は、ポリフエノール含量で示している。  FIG. 2 is a graph showing the hemagglutination-inhibiting action of the hop pellet cold water extract and the bent hop cold water extract on the HA titer of influenza virus. The vertical axis represents the HA value, and the concentration of each test extract is represented by the polyphenol content.
[0048] その結果、ホップペレット冷水抽出物及びスベントホップ冷水抽出物は、 0. 6mg/ mLの濃度でインフルエンザウイルスの HA価を有意に抑制した。この結果より、ホッ プ組織の冷水抽出物には、インフルエンザウイルスの赤血球凝集反応を抑制する物 質が含まれていることが判明し、抗ウィルス活性があることが示唆された。  [0048] As a result, the hop pellet cold water extract and the scavenged hop cold water extract significantly suppressed the HA titer of influenza virus at a concentration of 0.6 mg / mL. From this result, it was found that the cold tissue extract of hop tissue contains a substance that suppresses the hemagglutination reaction of influenza virus, suggesting that it has antiviral activity.
[0049] (実施例 2)インフルエンザウイルスの MDCK細胞への感染に及ぼすホップ組織の 冷水抽出物の効果：  [Example 2] Effect of cold water extract of hop tissue on infection of influenza virus to MDCK cells:
ホップ組織の冷水抽出物力 インフルエンザウイルスの動物細胞への感染を阻害 するか否かについて、正常ィヌ腎臓由来上皮細胞である MDCK細胞（Madin— Da rby canine kidney cells)を用いて調べた。 MDCK細胞は、さまざまなウィルス の感染実験に適した細胞であり、インフルエンザウイルスの感染阻害作用の評価に 一般的に用いられる細胞である。  Cold water extractability of hop tissue Whether or not to inhibit the infection of animal cells with influenza virus was examined using MDCK cells (Madin-Darby canine kidney cells), which are normal Inu kidney-derived epithelial cells. MDCK cells are suitable for various virus infection experiments and are generally used for evaluating the infection inhibitory action of influenza virus.
[0050] 1) MDCK細胞の培養 [0050] 1) MDCK cell culture
MDCK細胞は、 37°C、 5%の CO濃度下（COインキュベータ中）、 5%ゥシ胎児  MDCK cells at 37 ° C, 5% CO concentration (in CO incubator)
2 2  twenty two
血清 (FBS)を含有する MEM培地中で培養し、週 2回の頻度で継代培養して維持し た。インフルエンザウイルスの感染実験には、 3 X 105細胞 ZmLに調製した MDCK 細胞の細胞懸濁液を 96ゥエルプレートに 200 μ Lずつ添カ卩し、 3日又は 4日間培養し て使用した。 The cells were cultured in MEM medium containing serum (FBS) and maintained by subculture twice a week. In an influenza virus infection experiment, a cell suspension of MDCK cells prepared in 3 × 10 5 cells ZmL was added to a 96-well plate at 200 μL, and cultured for 3 or 4 days.
[0051] 2)インフルエンザウイルスの動物細胞への感染阻害試験  [0051] 2) Inhibition test of influenza virus on animal cells
図 3は、インフルエンザウイルスの動物細胞への感染阻害試験の手順を簡易的に 示した図である。まず、 25 Lのインフルエンザウイルス希釈液（上記のインフルェン ザウィルス液を 5 X 104倍希釈）と各濃度のホップペレット冷水抽出物又はスペントホ ップ冷水抽出物とを 96ゥエルプレート中で混合し、室温で 1時間反応させた (以下、こ の反応液をウィルス反応液と呼ぶ。；)。その後、予め 96ゥエルプレートで 3日間又は 4 日間培養した上記 MDCK細胞力 MEM培地を吸引除去し、そこに 25 μ Lの上記 ウィルス反応液を添カ卩し、 37°Cの COインキュベータ中でさらに 1時間反応させた。 Fig. 3 is a simplified diagram showing the procedure for an infection inhibition test for animal cells of influenza virus. First, a 25 L influenza virus dilution (the above influenza The virus solution was diluted 5 × 10 4 times) and each concentration of hop pellet cold water extract or spent hop cold water extract was mixed in a 96-well plate and allowed to react at room temperature for 1 hour (hereinafter referred to as “this”). The reaction solution is called a virus reaction solution;). After that, the MDCK cell force MEM medium previously cultured for 3 days or 4 days in a 96-well plate is removed by aspiration, and 25 μL of the virus reaction solution is added to the medium, and it is added in a 37 ° C CO incubator. The reaction was further continued for 1 hour.
2  2
反応後、各ゥヱルに 200 Lの維持培地（0. 2%アルブミン、 5 gZmLァセチルトリ プシンを含む MEM培地）を添カ卩して 3日間培養し、この培養上清に鶏赤血球凝集 作用がある力否かについて調べた。  After the reaction, 200 l of maintenance medium (MEM medium containing 0.2% albumin and 5 gZmL acetyltilpsin) is added to each tool and cultured for 3 days. The culture supernatant has the ability to agglutinate chicken erythrocytes. I investigated whether or not.
[0052] すなわち、インフルエンザウイルスの MDCK細胞への感染が成立した場合には、 3 日間の培養期間中にウィルス粒子が複製され、培地中にウィルス粒子が放出される ため、この培養上清の鶏赤血球凝集作用の有無を調べれば、インフルエンザウィル スの MDCK細胞への感染の阻害の程度を調べることができる。  [0052] That is, when infection of MDCK cells with influenza virus is established, virus particles are replicated during the 3-day culture period, and virus particles are released into the medium. By examining the presence or absence of hemagglutination, the degree of inhibition of influenza virus infection of MDCK cells can be determined.
[0053] 鶏赤血球凝集作用の有無は、上述した鶏赤血球凝集価測定試験とほぼ同じ手順 で調べられる力 HA価を求めない点で相違する。具体的には、 96穴マイクロプレー トの各ゥエルに 50 μ Lのインフルエンザウイルス液（TCID 値 = 105· 8)と 50 μ Lの各 [0053] The presence or absence of chicken erythrocyte agglutination is different in that it does not determine the strength HA value that can be examined by the same procedure as the chicken erythrocyte aggregation titer measurement test described above. Specifically, 50 μL of influenza virus solution (TCID value = 10 5 · 8 ) and 50 μL of each well of a 96-well microplate
50  50
培養上清又は 0. 1%BSA'PBSとをカ卩えて混合し、室温で 1時間反応させ、これに 5 0 Lの 0. 5%赤血球浮遊液をカ卩えて室温で 1晚静置する。その後、赤血球凝集像 の有無を観察し、各群 (n=6)の培養上清ごとに赤血球凝集が阻害された割合を調 ベ、赤血球凝集阻害率として評価した。さらに、赤血球凝集阻害率と被検抽出物の 濃度との関係から 50%感染阻害濃度 (TCID )についても求め、インフルエンザウイ  Culture supernatant or 0.1% BSA'PBS is mixed and mixed, allowed to react at room temperature for 1 hour, and 50 L of 0.5% erythrocyte suspension is added to this and left at room temperature for 1 hour. . Thereafter, the presence or absence of erythrocyte aggregation images was observed, and the ratio of inhibition of erythrocyte aggregation was examined for each culture supernatant of each group (n = 6) and evaluated as the erythrocyte aggregation inhibition rate. In addition, the 50% infection inhibitory concentration (TCID) was obtained from the relationship between the hemagglutination inhibition rate and the concentration of the test extract.
50  50
ルスの感染阻害活性の指標とした。  It was used as an index of the infection-inhibiting activity of lupus.
[0054] 図 4は、インフルエンザウイルスの MDCK細胞への感染に及ぼすホップペレット冷 水抽出物及びスベントホップ冷水抽出物の阻害作用を示すグラフである。縦軸は、 鶏赤血球凝集阻害率 (％)を表し、各被検抽出物の濃度は、ポリフエノール含量で表 している。 [0054] FIG. 4 is a graph showing the inhibitory action of the hop pellet cold water extract and the bent hop cold water extract on the infection of MDCK cells with influenza virus. The vertical axis represents chicken hemagglutination inhibition rate (%), and the concentration of each test extract is represented by the polyphenol content.
[0055] その結果、ホップペレット冷水抽出物及びスベントホップ冷水抽出物は、インフルェ ンザウィルスと室温で 1時間反応させることにより、 MDCK細胞への感染及び増殖を 阻害することが判明した。さらに、スベントホップ冷水抽出物は、 50 /z gZmLの濃度 で 80%以上の感染を阻害するものであり、同じ濃度のホップペレット冷水抽出物より も強いものであった。 [0055] As a result, it was found that the hop pellet cold water extract and the bent hop cold water extract inhibit infection and proliferation of MDCK cells by reacting with influenza virus for 1 hour at room temperature. In addition, the benthic hop cold water extract has a concentration of 50 / z gZmL Inhibits more than 80% of infections and is stronger than hop pellet cold water extract at the same concentration.
[0056] また、スベントホップ冷水抽出物の TCID を算出すると、 28. 6 μ gZmLであった  [0056] The TCID of the subvent hop cold water extract was calculated to be 28.6 μgZmL.
50  50
[0057] これらの結果より、ホップ組織の冷水抽出物には抗ウィルス活性があり、スペントホ ップの冷水抽出物には、ホップペレットの冷水抽出物よりも強い抗ウィルス活性があ ることが示唆された。  [0057] These results suggest that the hop tissue cold water extract has antiviral activity, and the spent hop cold water extract has stronger antiviral activity than the hop pellet cold water extract. It was done.
[0058] (実施例 3)スベントホップ冷水抽出物中に含まれるフラボノイドの分析：  [Example 3] Analysis of flavonoids contained in the extract of cold bent hop water:
1) LC— MSによる分析  1) LC—MS analysis
スベントホップ冷水抽出物中に含まれるフラボノイド成分を調べるため、以下の条件 下で、スベントホップ冷水抽出物の LC MS分析を行った。  In order to investigate the flavonoid components contained in the extract of cold bent hop water, LC MS analysis of the extract of cold bent hop water was performed under the following conditions.
LC MSの分析条件：  LC MS analysis conditions:
'溶離液: A液 0. 05%TFA水溶液、 B液 ァセトニトリル  'Eluent: A solution 0.05% TFA aqueous solution, B solution acetonitrile
•グラジェント条件： 0〜16min、 B液 10%〜50%  • Gradient condition: 0 ~ 16min, B liquid 10% ~ 50%
•流量： 0. 2mL/ mm  • Flow rate: 0.2mL / mm
•カラム温度： 40°C  • Column temperature: 40 ° C
，カラム： Waters Symmetry C18 2. I X 150mm 3. 5 πι  , Column: Waters Symmetry C18 2. I X 150mm 3.5 5 πι
•マススペクトル：（SIR;mZzl97、 m/z211, m/z287, m/z303)  • Mass spectrum: (SIR; mZzl97, m / z211, m / z287, m / z303)
[0059] 図 5は、スベントホップ冷水抽出物中に含まれるフラボノイドを LC MS分析した結 果である。その結果、ァストラガリン、イソケルシトリン、ケルセチンマロニルダルコシド 、ケンフェロールルチノシド、ケンフエロールマロ-ルダルコシド及びルチン並びにフ 口ロアシルフエノン配糖体であるフロロイソブチロフエノンダルコシド、フロロ一 2—メチ ルブチ口フエノンダルコシド及びフロロイソバレロフエノンダルコシドが含まれているこ とが判明した。 [0059] Fig. 5 shows the results of LC MS analysis of flavonoids contained in the bent-hop chilled water extract. As a result, astragalin, isoquercitrin, quercetin malonyl darcoside, kaempferol rutinoside, kenferrol malo-aldarcoside and rutin, and the fluoroacylphenone glycoside, fluoroisobutyrophenone darcoside, fluoro-2-methylbutyrate. It was found to contain oral phenone dalcoside and fluoroisovalerophenone darcoside.
[0060] 2)スベントホップ冷水抽出物中に含まれるフラボノィドアグリコンの分析  [0060] 2) Analysis of flavonoid aglycone contained in the extract of cold bent hop water
まず、 400 μ Lのスベントホップ冷水抽出物（20mgZmL)に 2mLの 2N塩酸をカロ え、沸騰水浴中で 30分間加熱して加水分解した。その後、 SepPak C18 (Waters 社)カラムに吸着する画分を、 2mLのメタノールで溶出し、得られたメタノール溶出物 を HPLCで分析した。 First, 400 mL of the benthop hop cold water extract (20 mgZmL) was charged with 2 mL of 2N hydrochloric acid and hydrolyzed by heating in a boiling water bath for 30 minutes. Subsequently, the fraction adsorbed on the SepPak C18 (Waters) column was eluted with 2 mL of methanol, and the methanol eluate obtained Was analyzed by HPLC.
[0061] HPLC分析条件は以下の通りである。 [0061] The HPLC analysis conditions are as follows.
HPLC分析条件：  HPLC analysis conditions:
'溶離液: A液 10mMリン酸水溶液、 B液 ァセトニトリル  'Eluent: A liquid 10 mM phosphoric acid aqueous solution, B liquid acetonitrile
.グラジェント条件：0〜20min、： B液 20%— 50% ; 20〜30min、： B液 50%〜10 0% ; 30〜40min、 B液 100%  Gradient conditions: 0 to 20 min, B solution 20% —50%; 20 to 30 min, B solution 50% to 100%; 30 to 40 min, B solution 100%
•流量： 0. 2mL/ mm  • Flow rate: 0.2mL / mm
•カラム温度： 40°C  • Column temperature: 40 ° C
，カラム： Waters Symmetry C18 2. I X 150mm 3. 5 πι  , Column: Waters Symmetry C18 2. I X 150mm 3.5 5 πι
，検出： UV370nm  , Detection: UV370nm
[0062] 表 2は、スベントホップ冷水抽出物の加水分解の前後におけるケルセチン及びケン フエロールの含有割合（％;ホップ冷水抽出物の乾燥重量重に対する各フラボノイド ァグリコンの重量比）を比較した結果を示したものである。  [0062] Table 2 shows the results of comparing the content ratios of quercetin and kaempferol before and after hydrolysis of the subvent hop cold water extract (%; weight ratio of each flavonoid aglycone to the dry weight of the hop cold water extract). Is.
[0063] [表 1]  [0063] [Table 1]
Figure imgf000015_0001
Figure imgf000015_0001
ND:検出限度以下  ND: Below detection limit
[0064] その結果、加水分解前では、ケルセチン及びケンフエロールを含むフラボノィドアグ リコンは検出されな力つた力 加水分解後では、ケルセチンが 1. 02%、ケンフェロー ルが 1. 21%含有されていることが判明した。加水分解前のスベントホップ冷水抽出 物には、フラボノィドアグリコンは検出されな力つたことより、スベントホップ冷水抽出 物中に含有されるフラボノイドは配糖体として存在し、抗ウィルス活性に寄与して!/ヽる ことが示唆された。 [0064] As a result, the flavonoid aglycone containing quercetin and kaempferol was not detected before hydrolysis. After hydrolysis, it contained 1.02% quercetin and 1.21% kaempferol. There was found. Since the flavonoid aglycone was not detected in the Sventhop cold water extract before hydrolysis, the flavonoids contained in the Sventhop cold water extract exist as glycosides, contributing to antiviral activity! / It was suggested to speak.
[0065] (実施例 4)インフルエンザウイルスのマウスへの感染に及ぼすホップ組織の冷水抽 出物の効果：  [Example 4] Effect of cold water extract of hop tissue on infection of influenza virus in mice:
ホップ組織の冷水抽出物力 インフルエンザウイルスのマウスへの感染を阻害する 力否力〖こついて、 BALBZcマウスとインフルエンザウイルスを使用した in vivoの感 染実験で調べた。 Cold water extractability of hop tissue Inhibits influenza virus infection in mice The inability to study force was investigated in an in vivo infection experiment using BALBZc mice and influenza virus.
[0066] マウス（BALBZcマウス、 7週齢、雌性）は、 日本エスエルシー（株）力 購入し、 1 週間の検疫期間を設けて予備飼育した。予備飼育で異常の認められな力つたマウス は、体重を測定し、各群の平均体重及び分散がほぼ等しくなるように無作為に 3群（ ホップペレット冷水抽出物投与群、スベントホップ冷水抽出物投与群及び対照群； 1 群 = 5匹）に分けた。  [0066] Mice (BALBZc mice, 7 weeks old, female) were purchased from Japan SLC Co., Ltd. and preliminarily bred with a one-week quarantine period. Mice that did not show any abnormalities in pre-bred animals were weighed and randomly divided into 3 groups (hop pellet cold water extract administration group, subvent hop cold water extract administration group) so that the average body weight and variance of each group were almost equal. Group and control group; 1 group = 5 animals).
[0067] ホップペレット冷水抽出物投与群は、ホップペレット冷水抽出物とインフルエンザゥ ィルスとを含む 0. 1%BSA'PBS溶液（ホップペレット冷水抽出物投与液）を、スペン トホップ冷水抽出物投与群は、スベントホップ冷水抽出物とインフルエンザウイルスと を含む 0. 1%BSA'PBS溶液 (スベントホップ冷水抽出物投与液)を、対照群はイン フルェンザウィルスのみを含む 0. 1%BSA'PBS溶液（対照用インフルエンザウィル ス投与液)を、各マウスの鼻孔に投与した。  [0067] The hop pellet cold water extract administration group contains a 0.1% BSA'PBS solution (hop pellet cold water extract administration solution) containing a hop pellet cold water extract and influenza virus, 0.1% BSA'PBS solution (svent hop cold water extract administration solution) containing Sventhop cold water extract and influenza virus, and the control group containing only influenza virus 0.1% BSA'PBS solution ( A control influenza virus administration solution) was administered to the nostrils of each mouse.
[0068] インフルエンザウイルスは、実施例 1で使用した A型 Puerto Rico/8/34 (PR8： H1N1)株を使用し、このインフルエンザウイルス株の原液 (TCID 値 = 105· 9)を 0. As the influenza virus, the type A Puerto Rico / 8/34 (PR8: H1N1) strain used in Example 1 was used, and the stock solution of this influenza virus strain (TCID value = 10 5 · 9 ) was set to 0.
50  50
1%BSAZPBSで 10倍希釈して調製したインフルエンザウイルス液を実験に用いた  Influenza virus solution prepared by diluting 10 times with 1% BSAZPBS was used for the experiment.
[0069] ホップペレット冷水抽出物及びスベントホップ冷水抽出物は、上述したように調製し 、ポリフエノール濃度が 50mgZmLとなるように 0. 1%BSAZPBSで溶解して用い た。 [0069] The hop pellet cold water extract and the scavent hop cold water extract were prepared as described above, and dissolved in 0.1% BSAZPBS so that the polyphenol concentration was 50 mgZmL.
[0070] マウスへの感染に使用するホップペレット冷水抽出物投与液及びスベントホップ冷 水抽出物投与液は、それぞれ 1. 98mLのホップペレット冷水抽出物及びスペントホ ップ冷水抽出物に上記のインフルエンザウイルス液をそれぞれ 0. 02mLずつ加えて 37°Cで 3分間反応させて調製した。対照用インフルエンザウイルス投与液につ！、て は、ホップ組織の冷水抽出物を含まない 0. 1%BSAZPBSに上記のインフルェン ザウィルス液を 0. 02mLカ卩えて 37°Cで 3分間反応させて調製した。  [0070] The hop pellet cold water extract administration solution and the subvent hop cold water extract administration solution used for infection of mice are 1. 98 mL of the hop pellet cold water extract and the spent hop cold water extract, respectively, and the above influenza virus solution. Were added at 0.02 mL each and reacted at 37 ° C for 3 minutes. A control solution for influenza virus! The above influenza virus solution was added to 0.1% BSAZPBS containing no hop tissue cold water extract and reacted at 37 ° C for 3 minutes.
[0071] マウスへの感染実験は、以下の手順で行った。まず、ネンブタールを腹腔内投与し てマウスを麻酔し、各群のマウスの両鼻孔にホップペレット冷水抽出物投与液、スぺ ントホップ冷水抽出物投与液又は対照用インフルエンザウイルス投与液を 10 μ Lず つ（20 μ LZマウス)投与した。投与開始から 2週間目まで連日、マウスの体重の推 移を記録すると共に毛並みの乱れ及び運動性の低下を観察し、インフルエンザウイ ルスの感染の有無及び生存数を調べた。インフルエンザウイルスの感染の有無は、 マウスの体重の推移、毛並みの乱れ及び運動性の低下を指標にし、 2項目以上が該 当する場合に感染成立と判断した。 [0071] Experiments on infection of mice were performed according to the following procedure. First, Nembutal was administered intraperitoneally to anesthetize the mice, and a hop pellet cold water extract administration solution and a spout were placed in both nostrils of each group of mice. 10 μL each (20 μLZ mouse) of the administration solution of the top hop cold water extract or the control influenza virus was administered. Every day from the start of administration, the changes in body weight of mice were recorded and observed for irregularities in hair and decreased motility, and the presence or absence of influenza virus infection and the number of survivors were examined. The presence or absence of influenza virus infection was determined to be established when two or more items were met, based on changes in mouse body weight, irregularities in hair, and decreased mobility.
[0072] なお、スベントホップ冷水抽出物投与群及びホップペレット冷水抽出物投与群と対 照群との間における感染率及び生存率の有意差の有無は、ログランク検定により解 祈した。 [0072] Whether or not there was a significant difference in the infection rate and survival rate between the administration group of the bent hop cold water extract and the administration group of the hop pellet cold water extract and the control group was reconsidered by the log rank test.
[0073] 図 6は、各群のマウスへのインフルエンザウイルスの感染率を、図 7は、各群のマウ スの生存率を示したものである。感染率が低ぐ生存率が高いほど、インフルエンザゥ ィルスのマウスへの感染が抑制されたことを意味している。  [0073] FIG. 6 shows the infection rate of influenza virus in mice in each group, and FIG. 7 shows the survival rate of mice in each group. The lower the infection rate and the higher the survival rate, the more the influenza virus infection was suppressed.
[0074] その結果、対照群では、投与開始 3日目に全てのマウスでインフルエンザウイルス の感染が認められ、投与開始 8日目に最初の死亡が確認され、投与開始 12日目に 全てのマウスの死亡が確認された。  [0074] As a result, in the control group, all mice were infected with influenza virus on the 3rd day from the start of administration, the first death was confirmed on the 8th day after the start of administration, and all mice on the 12th day after the start of administration. Was confirmed dead.
[0075] 一方、ホップペレット冷水抽出物投与群では、試験期間の 2週間にわたって、インフ ルェンザウィルスの感染は一匹も認められず、生存率についても 100%であった。ホ ップペレット冷水抽出物投与群の生存率は、対照群と比較して顕著に高ぐ両群の 間には統計的有意差 (ρ<0. 01)が認められた。  [0075] On the other hand, in the hop pellet cold water extract administration group, no infection with influenza virus was observed over the 2 weeks of the study period, and the survival rate was 100%. The survival rate of the hop pellet cold water extract group was significantly higher than that of the control group, and a statistically significant difference (ρ <0.01) was observed between the two groups.
[0076] また、スベントペレット冷水抽出物投与群では、投与開始 3日目に 2匹、投与開始 8 日目に全てのマウスにインフルエンザウイルスの感染が認められ、投与開始 10日目 には最初の死亡が確認された力 対照群のように全てのマウスが死亡することはなく 、 2週間経過後においても 3匹のマウスが生存していた。スベントペレット冷水抽出物 投与群は、対照群と比較して高い生存率を示し、両群の間には統計的有意差が認 められた（Ρく 0. 05)。  [0076] In addition, in the group administered with the cold pellet extract, 2 mice were observed on the third day of administration, and all mice were infected with influenza virus on the eighth day of administration. It was confirmed that the death of all mice did not die as in the control group, and 3 mice were alive even after 2 weeks. The group treated with the cold pellet extract showed a higher survival rate compared to the control group, and a statistically significant difference was observed between the two groups (Ρ 0. 05).
[0077] 以上の結果より、ホップペレット冷水抽出物及びスベントホップ冷水抽出物は、イン フルェンザウィルス感染を効果的に抑制することが示唆された。 産業上の利用可能性 本発明によれば、ウィルスの鶏血小板凝集反応を抑制する活性を有し、病原性ウイ ルスのヒト及び動物への感染を防止できる抗ウィルス剤が提供される。本発明の抗ゥ ィルス剤は、天然の植物由来であるため安全性に優れ、また、ビール等発泡性アル コール飲料の醸造の際の副産物として産出され、廃棄されるホップを原料とするため 、産業廃棄物の減少に貢献し、ホップの付加価値を高めて有効利用できる。 [0077] From the above results, it was suggested that the hop pellet cold water extract and the bent hop cold water extract effectively inhibit influenza virus infection. Industrial applicability ADVANTAGE OF THE INVENTION According to this invention, it has the activity which suppresses the chicken platelet aggregation reaction of a virus, and the antiviral agent which can prevent the infection to the human and animal of pathogenic virus is provided. Since the antiviral agent of the present invention is derived from a natural plant, it is excellent in safety, and since it is produced as a by-product when brewing foaming alcoholic beverages such as beer and discarded, hops are used as raw materials. It contributes to the reduction of industrial waste and can be used effectively with increased added value of hops.

Claims

請求の範囲 The scope of the claims
[I] ホップ組織の冷水抽出物を有効成分とする抗ウィルス剤。  [I] An antiviral agent comprising a cold water extract of hop tissue as an active ingredient.
[2] 前記ホップ組織は、乾燥されたホップ苞の粉砕物である、請求項 1記載の抗ウィル ス剤。  [2] The anti-virus agent according to claim 1, wherein the hop structure is a pulverized product of dried hop koji.
[3] 前記ホップ組織は、乾燥されたホップ毬花の粉砕物から、ルブリンの大きさ以下の 粉砕物の少なくとも一部が除かれたものである、請求項 1記載の抗ウィルス剤。  [3] The antiviral agent according to claim 1, wherein the hop tissue is obtained by removing at least a part of a pulverized product having a size smaller than that of rubulin from a pulverized product of dried hop spikelets.
[4] 前記乾燥されたホップ毬花の粉砕物は、乾燥されたホップ毬花の凍結物の粉砕物 である、請求項 3記載の抗ウィルス剤。 4. The antiviral agent according to claim 3, wherein the dried hop spikelet pulverized product is a dried hop spikelet frozen product pulverized product.
[5] 前記ホップ組織は、乾燥されたホップ毬花から、有機溶媒抽出又は超臨界流体抽 出される物質の少なくとも一部が除かれたホップ残渣である、請求項 1記載の抗ウイ ルス剤。 [5] The antiviral agent according to claim 1, wherein the hop tissue is a hop residue obtained by removing at least a part of a substance extracted from an organic solvent or a supercritical fluid from dried hop spikelets.
[6] ァストラガリン、ァストラガリンマロ-ルダルコシド、イソケルシトリン、イソケルシトリン マロ-ルダルコシド、ケルセチンマロ-ルダルコシド、ケンフェロールルチノシド、ケン フエロールマロ-ルダルコシド、ルチン及びフロロァシルフヱノン配糖体からなる群よ り選ばれるフラボノイド配糖体の少なくとも一つを含有する、請求項 1〜5のいずれか 一項記載の抗ウィルス剤。  [6] Consists of astragalin, astragalin malo-aldarcoside, isoquercitrin, isoquercitrin malo-aldarcoside, quercetin malo-aldarcoside, kaempferol rutinoside, kenferrol malo-aldarcoside, rutin and fluorosilacone glycoside The antiviral agent according to any one of claims 1 to 5, comprising at least one flavonoid glycoside selected from the group.
[7] 抗インフルエンザウイルス剤である、請求項 1〜6のいずれか一項記載の抗ウィルス 剤。  [7] The antiviral agent according to any one of claims 1 to 6, which is an anti-influenza virus agent.
[8] 請求項 1〜7の!、ずれか一項記載の抗ウィルス剤を含有する飲食品。  [8] A food or drink containing the antiviral agent according to any one of Claims 1 to 7!
[9] 請求項 1〜7の！、ずれか一項記載の抗ウィルス剤を有効成分とする医薬品。  [9] Claims 1-7! A pharmaceutical comprising the antiviral agent according to any one of the above as an active ingredient.
[10] 請求項 1〜7の!ヽずれか一項記載の抗ウィルス剤を含有する飼料。  [10] A feed containing the antiviral agent according to any one of claims 1 to 7!
[I I] 請求項 1〜7の!ヽずれか一項記載の抗ウィルス剤を含有する飼料添加物。  [I I] A feed additive containing the antiviral agent according to any one of claims 1 to 7.
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