JP2019104686A - Anti-influenza virus agent and composition containing the same - Google Patents

Abstract

To provide a novel anti-influenza virus agent and a composition containing the same.SOLUTION: An anti-influenza virus agent contains proanthocyanidin-containing plant extract, propolis extract, and optionally, lactic acid bacteria.SELECTED DRAWING: None

Description

  The present invention relates to an anti-influenza virus agent and a composition containing the same.

  Influenza is an acute respiratory infection that causes influenza virus as a pathogen, and different types of influenza are prevalent worldwide every year. In general, against influenza virus infection, prophylaxis by vaccination and treatment with drugs such as neuraminidase inhibitor are performed, but there is a problem that their effects differ significantly depending on the type of influenza virus. . In addition, depending on the drug, problems such as side effects and emergence of resistant virus remain.

  In view of the above problems, in the search for substances effective for the prevention and treatment of influenza virus, a safe and reliable anti-influenza virus agent that is less harmful to the human body, which is blended in food and drink, is desired. There is.

  Cranberry (Vaccinium macrocarpon Ait.) Is a plant of the azalea genus Vaccinium vulgaris which grows in cold regions such as North America, and produces small fruits. In the United States, breed improvement is carried out and is grown on a large scale. Cranberry fruit can be eaten as it is, but it has been widely used as dried fruit and as a beverage, juice source, cooking sauce, jam, jelly confectionery and so on. In addition, intake of cranberry juice is known to be effective for urinary tract infections.

  Propolis is a resinous material that bees store in hive boxes, which contain resin, beeswax, essential oils, pollen, flavonoids, etc. It is used for promotion.

Planta Med. 2012; 78 (10): 962-7 Antiviral Res. 2005; 66 (1): 9-12 Nutr J. 2013; 12: 161 J Biomol Screen. 2012; 17 (5): 605-17 Antivir Chem Chemother. 2008; 19 (1): 7-13

Unexamined-Japanese-Patent No. 2011-168572

  An object of the present invention is to provide a novel anti-influenza virus agent and a composition containing the same.

  There are various extracts derived from natural products having anti-influenza virus action, for example, the extract of cranberry is combined with the extract obtained by extracting and treating the fruiting body and / or mycelium of Agaricus moth with a hydrophilic solvent. It is known to significantly suppress influenza virus infection and / or growth in vivo after the infection (Patent Document 1). In addition, it is also known that cranberry extract alone or proanthocyanidin which is an antioxidant component contained in cranberry inhibits the growth of various viruses such as influenza virus (Non-patent documents 1-4).

  Also with regard to propolis, there is a finding that its ethanol extract is effective against influenza infection (Non-patent Document 5).

  In addition, various natural product extracts have been reported to have anti-influenza virus action, but the effect is considered to be insufficient. In fact, as if to support this, none of the extracts have generally been used in Japan for influenza control.

  When the present inventors confirmed the effect of a component known to have anti-influenza virus action among a large number of components that can be incorporated into food and drink, the anti-influenza virus action alone is not so strong. It became clear that the combination may increase the effect. As a result of further studies on these specific combinations, the present inventors found that (A) proanthocyanidin-containing plant extract, (B) propolis extract, and, optionally, (C) a composition containing lactic acid bacteria ( A), (B), and (C) were found to have an anti-influenza effect far exceeding the sum of the effects alone, leading to the present invention.

The summary of the present invention is as follows.
(1) An anti-influenza virus agent comprising proanthocyanidin-containing plant extract and propolis extract as active ingredients.
(2) The anti-influenza virus agent as described in (1) whose proanthocyanidin containing plant is a plant which belongs to berries.
(3) The anti-influenza virus agent as described in (1) or (2) whose plant which belongs to berries is cranberry.
(4) The anti-influenza virus agent in any one of (1)-(3) by extraction of an extract with ethanol.
(5) Any one of (1) to (4) according to any of (1) to (4), further comprising lactic acid bacteria or other fungi having interleukin-12 (IL-12) production promoting action or containing double-stranded RNA Anti-influenza virus agent.
(6) The anti-influenza virus agent as described in (5) whose lactic acid bacteria are lactic acid bacteria which belong to Lactobacillus paracasei (Lactobacillus paracasei).
(7) The anti-influenza virus agent according to (6), wherein Lactobacillus paracasei is a Lactobacillus paracasei MCC 1849 (NITE BP-01633) strain.
(8) A composition comprising the anti-influenza virus agent according to any one of (1) to (7).
(9) The composition according to (8), which is blended in food and drink, a supplement or a medicine.
(10) The composition according to (8) for the prevention or treatment of influenza virus infection.
(11) 0.1 to 0.98 parts by weight of proanthocyanidin, 0.01 to 0.98 parts by weight of propolis extract, 0.01 to 0.98 parts by weight of lactic acid bacteria in terms of dry weight, (8) to The composition as described in any one of (10).

  The anti-influenza virus agent and the composition for food and drink of the present invention contain (A) proanthocyanidin-containing plant extract, (B) propolis extract, and, optionally, (C) lactic acid bacteria, and have excellent anti-influenza effects. In particular, the combination of extracts (A) and (B) has been confirmed to have a significant synergistic effect on anti-influenza virus action. Further, it is considered that any component can be safely taken orally since it is a component to be blended in food and drink.

  Hereinafter, embodiments of the present invention will be described. The present invention is not limited to the following embodiment.

(Anti-influenza virus agent)
The anti-influenza virus agent of this embodiment contains proanthocyanidin-containing plant extract and propolis extract as active ingredients. The anti-influenza virus action broadly means suppressing the activity of influenza virus, particularly influenza A virus. While not intending to be bound by theory, the anti-influenza virus agent of the present invention is expected to suppress the growth of influenza virus in the body, alleviate any symptoms caused by influenza virus, and prevent the seriousness. Be done. The anti-influenza virus agent of the present invention can be used not only for the alleviation of each symptom but also for the treatment of influenza and the prevention of secondary infection and the like.

(A) Proanthocyanidin-Containing Plant Extract Proanthocyanidin is a condensation type tannin present in various plants or non-hydrolyzable tannin, that is, flavan-3-ol or flavan-3,4-diol as a constituent unit, condensation, polymerization And a group of compounds bound by Proanthocyanidins are classified into several types due to the difference in binding mode, for example, two or more flavan-3-ol structures are 4β → 8 and 2β → O → 7 or 4β → 6 and 2β When there is at least one site that binds via position → O → 7, type A, and two or more flavan-3-ol structures are linked via 4β → 8 or 4β → 6 It is known that when it is done, it is classified into type B. Proanthocyanidin contained in the extract may be either A-type or B-type or both, but A-type is preferred.

  Proanthocyanidin-containing plants mean plants containing proanthocyanidin. Although not intended to be limiting, proanthocyanidin containing plants include berries, plums, avocado, peanuts, cinnamon and the like. Among the berries, cranberries known to be rich in proanthocyanidins are preferred.

  As used herein, a proanthocyanidin-containing plant extract is, for example, a juice obtained by squeezing juice from a site containing proanthocyanidin such as berry fruit or a concentrate of the juice, the juice or the concentrated juice Containing solids in the juice obtained by means of adding and mixing excipients such as dextrin and spray drying or lyophilizing, or a solvent that is more suitable than fruits, peels and seeds containing proanthocyanidin The extract etc. which were extracted by using it. The form of the extract is not important as long as it contains a predetermined amount of the active ingredient, and may be a soft extract or a dry extract or other similar forms such as a dry state. The same applies to the other extracts used in the present invention.

  The solvent used for the extraction treatment is not particularly limited, and conventional polar solvents, ambipolar solvents and the like can be used. As the solvent, for example, water, an organic solvent or a solvent containing it, a mixed solution of water and an organic solvent, and the like can be used. As the organic solvent, for example, lower alcohols (specifically ethanol, methanol, propanol, butanol), ethers (specifically diethyl ether), halogenated hydrocarbons (specifically chloroform), nitriles (specifically In addition to acetonitrile, esters (specifically, ethyl acetate), ketones (specifically, acetone), etc., hexane, dimethylsulfoxide, dimethylformamide, etc. are used, but from the viewpoint of workability, ethanol, Methanol or ethyl acetate is preferred. The organic solvent may be used as a mixture of two or more.

  When proanthocyanidin is extracted from a plant material, a method of extracting using an organic solvent such as water or ethanol, drying and concentrating (for example, JP-A-9-221484, re-table 2005/079825, re-table 2006/068212 And the like are known. By these methods, proanthocyanidin type B can be efficiently extracted from fruit skin and the like. For proanthocyanidin type A, in order to increase its extraction efficiency, a solution obtained after extraction is subjected to adsorption treatment with a synthetic adsorbent or a reverse phase adsorbent (for example, Japanese Patent No. 2975997, re-table 2005/030200 No. 2005/072762, Japanese Patent Laid-Open No. 2008-156265, and Japanese Patent Laid-Open No. 2008-156265, etc. are known. Further, a method is disclosed in Japanese Patent No. 5459699, which improves the problems associated with the extraction of proanthocyanidin A.

  In order to concentrate and purify the active ingredient in the cranberry extract, further solvent extraction, synthetic adsorbent treatment, ion exchange resin treatment, etc. may be performed.

  The compounding amount of the proanthocyanidin-containing plant extract also varies depending on the desired effect and the amount of proanthocyanidin or other active ingredients contained in the extract, for example, when the content of the proanthocyanidin in the extract is 40% by mass, It may be about 0.05 to 0.98 parts by weight with respect to 100 parts by weight of the influenza virus agent. When considering the anti-influenza virus action alone, for example, assuming that the content of proanthocyanidin in the extract powder is 40% by mass, the compounding amount of the extract is about 5 to about 40 μg / final concentration in the anti-influenza virus agent It may be adjusted to be in the range of ml, preferably about 7.8 to about 31.3 μg / ml.

(B) Propolis Extract Propolis is a collection of viscous resins, gums and balsams, which are transported to the nest and present with their own salivation and addition of wax. Refers to a collection of viscous resins, gums and balsams that are collected on certain parts of the plant, in particular buds and barks, by a bee improving to some extent.

  The propolis may be mixed as a carrier with an excipient such as maltodextrin, a hydroalcoholic solution. The form of the extract is not limited to liquid, and may be powder. For example, a propolis extract obtained by ethanol extraction may be kneaded with dextrin and used as a propolis powder.

  The propolis extract refers to an extract of propolis (raw mass) by water, hydrous ethanol or ethanol, supercritical extraction method, etc. according to the propolis food voluntary standard standards of the Japan Propolis Council. In the present specification, propolis extract in “Process of processed propolis extract” (propolis extract content of 10% or more) of Japan Health and Nutrition Foods Association is used. The amount of propolis extract described herein is expressed according to the calculation method described in the Propolis Food Standard Specification (April 2010) unless otherwise specified. The details will be described below.

  From the high performance liquid chromatogram (HPLC) of the test solution, determine the total area of all peaks of the retention time up to 30 minutes (up to 45 minutes retention time when using water-extracted propolis extract). Next, the peak area of the p-coumaric acid standard solution is determined and used as a standard area. The total area of all peaks is regarded as the amount corresponding to p-coumaric acid, and the corresponding amount is calculated. The amount of propolis soluble component is calculated from this p-coumaric acid equivalent using a conversion factor. Finally, it is determined that the propolis soluble component amount is equal to or greater than the indicated value.

  The compounding amount of propolis extract powder also varies depending on the desired effect and the amount of solid content derived from propolis or other active ingredients contained in the extract, for example, using a raw material containing 25% as p-coumaric acid equivalent amount If it is, it may be about 0.01 to 0.98 parts by weight with respect to the anti-influenza virus agent. When considering the anti-influenza virus action alone, for example, using a material containing 25% as p-coumaric acid equivalent, the final concentration is about 200 to about 500 μg / ml, preferably about 250 to about 300 μg / ml The range can be adjusted.

(C) Lactic Acid Bacteria Lactic acid bacteria have been reported to exhibit preventive and protective effects against various infectious diseases. It has been reported that these actions of lactic acid bacteria are due to activation of cellular immunity of the host and promotion of secretion of IgA from mucous membranes such as the intestinal tract and respiratory organs. Lactic acid bacteria belonging to Lactobacillus casei can be used as a host by inducing the production of cytokines such as IL-12 (interleukin-12) and IFN-γ (interferon-γ) from host immunocompetent cells. It has been reported that cellular immunity is activated to prevent infection with influenza virus and the like.

  The anti-influenza virus action exerted by the lactic acid bacteria in the present invention may be different from the above extract. Therefore, even if the anti-influenza virus activity in vitro is low, any lactic acid bacteria that significantly increase cytokine production can be suitably used in the present invention. For example, double-stranded RNA is known to promote cytokine production (Japanese Patent No. 5099649), and a lactic acid bacterium containing a large amount of double-stranded RNA is considered to be preferable for combination with the above extract.

  The content of double-stranded RNA in the lactic acid bacteria of one embodiment of the present invention is the content of double-stranded RNA of the strain when the same strain is cultured under the optimal temperature and non-aerated conditions for the same culture time. The amount is 2.0 times or more, preferably 3.0 times or more, more preferably 5.0 times or more, and still more preferably 10 times or more. Preferably, the amount may be 30 times or more. Moreover, it is preferable that lactic acid bacteria of 1 aspect of this invention are lactic acid bacteria whose content of double stranded RNA is 1,000 ng / mL or more per 5 mg of dry cell bodies.

  Lactic acid bacteria having IL-12 production promoting activity or containing double stranded RNA can be obtained by subjecting the lactic acid bacteria to culture under predetermined conditions. The lactic acid bacteria used in this case mean microorganisms which produce lactic acid as generally known. Examples of the lactic acid bacteria include, for example, Pediococcus bacteria, Lactococcus bacteria, Lactobacillus bacteria, Lactobacillus bacteria, Streptococcus bacteria, Leuconostoc bacteria, Tetragenococcus Lactic acid bacteria belonging to the genus of lactic acid bacteria (Tetragenococcus) and the like are mentioned. Specifically, Pediococcus acidilactici, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus Bacillus pentosus (Lact bacillus pentosus, Lactobacillus sakei, Tetragenococcus halophilus, Pediococcus pentosaceus, Lactobacillus delbrucky subspecies bulgaricus (Lactobacillus delbrubeii sp.) bulgaricus, Lactobacillus brevis, Lactobacillus casei subsp. casei, Lactobacillus paracasei subsp. paraca B. (Lactobacillus paracasei subsp. Paracasei), Streptococcus thermophilus (Streptococcus thermophilus), Leuconostoc mesenteroides subsp. Mesenteroides (Leuconostoc mesenteroides subsp. Mesenteroides), Leuconostoc mesenteroides Examples include, but are not limited to, Stock Lactis (Leuconostoc lactis).

  More specific non-limiting examples of lactic acid bacteria include Pediococcus acidilactici strain K15, Lactococcus lactis strain ATCC 19435, Lactobacillus plantarum strain ATCC 14917, Lactobacillus pentosus strain ATCC 8041, Lactobacillus strain Sakei K 41 strain, Tetragenococcus halophilus KK 221 strain, Tetragenococcus halophilus NBRC 12172 strain, Pediococcus pentosaceus OS strain (NITE P-354), Pediococcus pentosaceus NRIC 1915, Pediococcus pentosaceus NRIC 0099, Pediococcus pentosaceus NRIC0122 strain, Lactobacillus plantarum NRIC 1930 strain, lactobacilli -Plantarum NRIC 1067, Lactobacillus delbrucki subsp. Bulgaricus NRIC 1688, Lactobacillus delbrucky subsp. Lactis NRIC 1683, Lactobacillus brevis NRIC 1713, Lactobacillus pentosus NRIC 0391, Lacto B. pentosus NRIC 0396 strain, Lactobacillus pentosus NRIC 1836 strain, Lactobacillus casei subspecies casei NRIC 0644 strain, Lactobacillus paracasei subspecies paracasei NRIC 1936, Streptococcus thermophilus strain NRIC 0256, Leuconostoc mesenteripeides species・ Messenteroide NRIC1982 share, Leuconostoc shoe domain initiative Roy Death ATCC12291 strain, and the like Leuconostoc lactis NRIC1582 strain.

  As lactic acid bacteria used to obtain the lactic acid bacteria of one embodiment of the present invention, one of the above-described ones can be used alone, or two or more kinds can be used in combination. Further, among the above, from the viewpoint of double-stranded RNA content, Pediococcus acidilactici K15 strain, Lactococcus lactis ATCC 19435 strain, Lactobacillus plantarum strain ATCC 14917 strain, Lactobacillus pentosus strain ATCC 8041 And Lactobacillus sakei strain K41 is preferred, and Pediococcus acidilactici strain K15 is more preferred.

  In another preferred embodiment, the lactic acid bacteria may be Lactobacillus paracasei MCC 1849 (NITE BP-01633) strain, which is a novel strain of lactic acid bacteria belonging to Lactobacillus paracasei (Patent No. 5646124) .

  Lactobacillus paracasei used in the present invention is not limited to the above-mentioned deposited strain, and may be a strain substantially equivalent to the deposited strain. A substantially equivalent strain is a strain belonging to Lactobacillus paracasei, which has an action of promoting IL-12 production as high as that of the deposited strain, and is preferably treated with a cell wall degrading enzyme and RNase Also refers to strains with as little reduction in IL-12 production promoting activity as deposited strains. Further, the substantially equivalent strain is further such that the base sequence of its 16S rRNA gene is 98% or more, preferably 99% or more, more preferably 100% homologous to the base sequence of the 16S rRNA gene of the deposited strain. And preferably have the same mycological properties as the deposited strain. Furthermore, the lactic acid bacteria of the present invention may be a strain bred from a deposited strain, or a strain substantially equivalent thereto, by mutation treatment, genetic recombination, selection of a naturally occurring mutant, etc., as long as the effects of the present invention are not impaired. It may be.

  The compounding amount of the lactic acid bacteria also varies depending on the desired effect, the kind of lactic acid bacteria or the amount of other active ingredients, etc. It may be about 0.98 parts by weight (dry weight conversion). When considering the anti-influenza virus action alone, for example, the final concentration of Lactobacillus paracasei MCC 1849 is in the range of about 200 to about 1,500 μg / ml, preferably about 250 to about 1,000 μg / ml on a dry weight basis. The blending amount can be adjusted to be

(Composition)
The composition of the present embodiment contains the above anti-influenza virus agent. The composition of the present invention may be formulated into food and drink, supplements or medicines.

  The blending ratio (mass ratio) of the total amount of (A) cranberry extract, (B) propolis extract, and (C) lactic acid bacteria in the composition of the present invention is not particularly limited to the amounts described for each component, and the purpose and use It can be set appropriately according to the conditions of the object etc.

  The composition of the present invention may contain other components in addition to (A) cranberry extract, (B) propolis extract, and (C) lactic acid bacteria. As other components, for example, proteins, dietary fibers such as water-soluble dietary fibers, insoluble dietary fibers, minerals, processed plants or processed plant products, algae, microorganisms other than lactic acid bacteria, etc. can be blended.

  Furthermore, as necessary, dextrin, saccharides such as starch, oligosaccharides, sweeteners, acidulants, colorants, thickeners, brighteners, excipients, vitamins, nutritional supplements, which are usually used in the food and beverage field, A binder, a lubricant, a stabilizer, a diluent, an extender, an emulsifier, a food additive, a seasoning, etc. can be mentioned. The content of these other components can be appropriately selected according to the form of the composition of the present invention and the like. In addition, depending on the purpose of the final product, additional components may be added to the composition as appropriate.

  The form of the composition of the present invention is not particularly limited, and may be any form. As a specific form, for example, a powder, such as powder, granules, powder such as fine granules, a tablet (chewable), a sphere, a capsule, a caplet, a liquid or the like can be mentioned. The capsule-like composition includes soft capsules and hard capsules.

  The composition of the present invention is preferably in the form of powder, such as powder, granules, fine particles, etc., and in particular, it is mixed with water or the like and dissolved or suspended to form a powdered beverage to be used. It is preferable because it is excellent in stability and can take many compositions at a time unlike capsules and tablets.

  The composition of the present invention can be produced by a conventionally known method. When manufacturing the composition of this invention, the form of the raw material to be used is not specifically limited, According to the form of a composition, it can select suitably and can be used. For example, in the case of obtaining a powdery composition such as powder, granules or fine granules, (A) cranberry extract, (B) propolis extract and (C) lactic acid bacteria may be used as they are, or an excipient And mixtures with bulking agents and the like may be used. Moreover, when obtaining a capsule-like composition, you may use what was melt | dissolved or disperse | distributed beforehand in solvent, such as water and an edible oil.

  Hereinafter, the present invention will be specifically described by way of examples. The present invention is not limited to these examples.

・ (In vitro anti-influenza virus activity evaluation test)
About (A) cranberry extract, (B) propolis extract, and (C) lactic acid bacteria (Lactobacillus paracasei MCC 1849) (hereinafter, three types may be collectively referred to as "test substance"), type A influenza virus (H1N1) The antiviral activity against was evaluated.

  The evaluation was carried out in an in vitro infection system using cultured cells, and the antiviral activity as well as the cytotoxicity of the test substance was evaluated. The test first evaluates antiviral activity and cytotoxicity when using (1) (A) cranberry extract, (B) propolis extract and (C) lactic acid bacteria alone, and after determining the test content based on the results, The antiviral activity was evaluated for the combined use of (2) (A) cranberry extract, (B) propolis and (C) lactic acid bacteria.

・ (Test material)
The raw materials (A) to (C) were all in powder form. Cranberry extract powder is 40% of total proanthocyanidin standard made by Nexilla Co., Ltd. Propolis extract powder is Brazilian propolis extract 20% powder (excipient: β-CD) manufactured by Morikawa Kushido Co., Ltd. (note: β- CD means (beta) -cyclodextrin), and the lactic acid bacteria end used shield lactic acid bacteria M-1 (Lactobacillus paracasei MCC1849 strain) by Morinaga Milk Industry Co., Ltd. respectively.

The control substances used are as follows.
Substance name: Zanamivir hydrate (trade name: Relenza (registered trademark))
Origin: GlaxoSmithKline Solvent: DMSO (Dimethyl sulfoxide)
Storage method: Storage at -20 ° C after dissolution Remarks: Neuraminidase inhibitors

The viruses used are as follows.
Virus name: influenza A virus strain name: WSN (A / WSN / 33, H1N1) strain origin: AVSS strain (control number: H1611), virus solution amplified in MDCK cells and recovered in culture supernatant

The cells used as infection hosts for influenza virus are as follows.
Cell line name MDCK (Madin-Darby Canine Kidney) cell-derived (dog kidney-derived cell line)

(Reagents and instruments)
MEM medium (Wako, Cat. # 051-07615)
-Fetal bovine serum FBS (Sigma, Cat. # 172012-500 ML)
100 × MEM vitamin (Gibco, Cat. # 11120-052)
・ Penicillin G potassium (nacalai, Cat. # 26239-42)
Streptomycin sulfate (nacalai, Cat. # 32204-92)
-Dimethyl sulfoxide (Wako, Cat. # 031-24051)
Trypsin (Thermo Fisher, Cat. # 15090046)
-96-well plate (TPP, Cat. # 92096)
・ Φ 10 cm dish (TPP, Cat. # 93100)
・ Microplate reader (TECAN, infinite M200)
・ Plate shaker (M & S, Microplate genie)
・ 8 series micro pipettes (GILSON)
・ Micro pipette (20, 200, 1000 L, eppendorf)

·(Test method)
The test was conducted in two stages of "Test (1)" and "Test (2)".
In the test (1), anti-influenza virus activity and cytotoxicity when each of the three test substances was used alone were evaluated.

  Next, in the test (2), based on the result of the test (1), the test concentration and plate layout of each test substance were determined, and then the antiviral activity of the three combinations was evaluated.

  The outline of the test (1) is shown below. The test (2) differs from the test (1) in the amount of liquid when adding each test substance, but the basic method is the same.

· (Method for preparing test substance)
For cranberry extract powder and propolis extract powder, these are weighed, ethanol is added to adjust the concentration to 100 mg / ml, infection maintenance medium (vitamin-containing serum free MEM) is added, and the concentration of each test substance is The sample was prepared to be 2 mg / ml. This gave a final concentration of 2% (v / v) of ethanol used as solvent. About the lactic acid bacteria end, it adjusted so that it might become 2 mg / ml in the said infection maintenance medium. The prepared samples were stored at 4 ° C. until use. On a 96-well plate separately prepared for dilution, a total of 10 concentrations (excluding non-added groups) were prepared in 2-fold serial dilution series with 2 mg / ml as the highest concentration. The infection maintenance medium was used for dilution. Also, a 2-fold serial dilution series of positive control substances was prepared in the same manner.

・ (Anti-influenza virus activity)
MDCK cells were prepared at 3.0 × 10 ⁵ cells / ml with 10% serum-containing MEM (MEM-10% FBS) and seeded at 100 μl / well onto 96-well plates (eg 3.0 × 10 ⁴ cells) / Well).
However, no cells were seeded on the first row of the plate at the first row (total of 8 wells), and this was used as a correction blank group at the time of analysis. After culturing for 24 hours at 37 ° C. under 5% CO ₂ , the culture medium was removed, and the cell monolayer of each well was washed once with 100 μl / well of serum free MEM.

After washing, add 2-fold serial dilution series such as test substance to be prepared at 100 μl / well, and continue to add 100 μl of influenza virus solution prepared to 1,000 TCID ₅₀ / ml in the infection maintenance medium in the antiviral activity evaluation group / Well was added (MOI ≒ 0.002 pfu / cell equivalent).

The test substance etc. was diluted 2-fold from each concentration of 0-2 mg / ml, and adjusted to the final test concentration shown to 0-1 mg / ml. After virus addition, the 96-well plate was stirred at room temperature for 30 seconds, and then cultured at 37 ° C. under 5% CO ₂ for 3 days (72 hrs). In addition, the test was implemented by N = 2 (duplicate).

・ (Method for quantifying and analyzing the results)
The culture solution was removed from the 96-well plate after culture, and after fixing the cells with a 70% aqueous ethanol solution (5 minutes at room temperature), the ethanol was removed and the remaining cells were stained with a 0.5% aqueous crystal violet solution. After staining, the plate was lightly rinsed with tap water and allowed to air dry.

After air-drying, the absorbance (λ = 560 nm) of each well was measured with a microplate reader, and based on the obtained measurement results, 50 using a statistical analysis software “GraphPad Prism 5.0 (GraphPad Software)”. The% infection inhibitory concentration (IC ₅₀ ) was calculated.
The results are shown below.
Table 1: Anti-influenza virus activity when used alone

  As a result, when the cranberry extract was added alone, a concentration-dependent increase in antiviral activity was observed at an additive concentration of 7.8 to 31.3 μg / ml. Propolis extract alone showed remarkable antiviral activity when the added concentration was around 250 μg / ml. Lactic acid bacteria powder alone also showed an increase in antiviral activity at a concentration of 250 to 1,000 μg / ml.

Furthermore, when all the components of (A) to (C) were combined and the change in antiviral activity of cranberry extract was observed, it was possible to confirm a remarkable anti-influenza virus action significantly exceeding the effect of each component alone . Some of the results are shown below.
Table 2: Cranberry extract IC ₅₀ list at the time of combination of three test substances

(Production of composition for eating and drinking)
Soft capsules having the formulations shown in Table 3 below were prepared.

  According to the present invention, an excellent anti-influenza composition can be provided by containing cranberry extract, propolis extract, and, optionally, lactic acid bacteria.

Claims (11)

  An anti-influenza virus agent comprising proanthocyanidin-containing plant extract and propolis extract as active ingredients.   The anti-influenza virus agent according to claim 1, wherein the proanthocyanidin-containing plant is a plant belonging to berries.   The anti-influenza virus agent according to claim 1 or 2, wherein the plant belonging to the berry is cranberry.   The anti-influenza virus agent according to any one of claims 1 to 3, wherein the extract is extracted with ethanol.   The anti-influenza virus according to any one of claims 1 to 4, further comprising lactic acid bacteria or other fungi having interleukin-12 (IL-12) production promoting activity or containing double stranded RNA. Agent.   The anti-influenza virus agent according to claim 5, wherein the lactic acid bacterium is a lactic acid bacterium belonging to Lactobacillus paracasei.   The anti-influenza virus agent according to claim 6, wherein the Lactobacillus paracasei is a Lactobacillus paracasei MCC 1849 (NITE BP-01633) strain.   A composition comprising the anti-influenza virus agent according to any one of claims 1 to 7.   The composition according to claim 8, which is incorporated in a food or drink, a supplement or a medicine.   The composition according to claim 8, for the prevention or treatment of influenza virus infection.   The method according to any one of claims 8 to 10, comprising 0.1 to 0.98 parts by weight of proanthocyanidin, 0.01 to 0.98 parts by weight of propolis extract, and 0.01 to 0.98 parts by weight of lactic acid bacteria on a dry weight basis. The composition according to any one of the preceding claims.