KR20160090495A

KR20160090495A - Anti-influenza viral agent

Info

Publication number: KR20160090495A
Application number: KR1020150010337A
Authority: KR
Inventors: 류형준
Original assignee: 류형준
Priority date: 2015-01-22
Filing date: 2015-01-22
Publication date: 2016-08-01

Abstract

The present invention relates to an anti-influenza viral agent comprising manuka honey, propolis and xylitol, as active ingredients. The anti-influenza viral agent is a natural complex which has effects of inhibiting and treating influenza virus. In addition, the anti-influenza viral agent can be applied to a health drink, a cold treatment tea, a rhinitis spray, a cold preventing soap, a cold preventing detergent, and the like.

Description

Anti-influenza virus (ANTI-INFLUENZA VIRAL AGENT)

The present invention relates to an anti-influenza virus agent, and more particularly, to an anti-influenza virus agent comprising manuka honey, propolis and xylitol as an active ingredient.

Influenza virus is an RNA virus belonging to the family Orthomyxoviridae, which causes inflammation in the respiratory tract. It is transmitted directly to the air by the cough and saliva of the infected person, or indirectly by the contact with influenza patients. , And the serotypes of the virus are divided into A type, B type and C type. Among them, types B and C have been confirmed to be infected only in humans. Type A infection has been confirmed in humans, horses, pigs, other mammals and various kinds of poultry and wild birds. The serotype of influenza A virus is classified according to the types of hemagglutinin (HA) and neuraminidase (NA), two proteins on the virus surface, and 144 types (HA type 16 species and 9 types of NA type). The HA protein is responsible for the attachment of the virus to the somatic cell, and the NA protein allows the virus to penetrate into the cell. Recently, a new type of influenza (H1N1) that is prevalent in the world is Human Influenza A type. Subtypes are HA (hemagglutinin) protein type 1 (H1) and NA (neuraminidase)

Influenza A (H1N1) virus.

Known as an antiviral agent for type A influenza virus, artificial synthetic zanamivir (trade name: Relenza) and oseltamivir (trade name: TAMIFLU) as a neuraminidase inhibitor of influenza virus have. Zanamivir and oseltamivir inhibit the function of the neuraminidase protein of influenza viruses and are typical antiviral agents that inhibit the growth of influenza viruses in vivo. Both antiviral agents are known to be effective against all 16 serotype A influenza viruses and serotype B influenza viruses. However, there is a disadvantage in that it is administered by inhalation and intravenous administration, and oseltamivir can be orally administered, but recent reports of the emergence of resistant virus and side effects such as vomiting and dizziness when administered orally have been pointed out. Accordingly, there have been various developments of anti-influenza virus agents that can be used in the form of drink, tea, spray, soap, detergent, feed additive, and the like.

Korean Patent No. 10-1118230 discloses a plant extract having an anti-influenza virus activity including a plant extract selected from one or more of Barnabas, Acanthopanax or Oregano as an example of a natural influenza virus-derived anti-influenza virus agent, 10-1426888 discloses an avian influenza virus proliferation inhibiting and anti-avian influenza virus composition comprising ginseng or red ginseng extract, saponin or an acid polysaccharide as an active ingredient, and Korean Patent No. 10-0923884 discloses a composition for inhibiting avian influenza virus Alnus japonica). Korean Patent No. 10-1035463 discloses a composition for preventing and treating highly pathogenic avian influenza virus comprising an extract extracted from a black ginseng root as an active ingredient. In Korean Patent No. 10-1322851, Korean Patent No. 10-1139376 discloses a composition for inhibiting the proliferation of avian influenza virus, which comprises a mixture of citrus spp., Licorice, and garlic. Myrica cerifera, Ruta graveolens, Helichrysum italicum, Tilia cordata, Pimenta officinalis, Theobroma cacao, Chaenomeles sinensis, Pseudocydonia sinensis Korean Patent Laid-Open Publication No. 10-2014-0104652 discloses a composition for anti-influenza comprising an extract of a scabbard flower as an active ingredient, Japanese Patent Registration No. 10-1443510 discloses a pharmaceutical composition for preventing or treating influenza, which contains an extract of Cichorium intybus as an active ingredient Korean Patent No. 10-1310033 discloses a composition for preventing or treating an influenza virus infection comprising an extract of Aspergillus oryzae as an active ingredient, Korean Patent Publication No. 10-2011-0093975 discloses a composition containing Turmeric extract Korean Patent No. 10-1376231 discloses a composition for the prevention and treatment of influenza virus comprising an extract of sprout barley as an active ingredient, and Korean Patent Publication No. 10-1376231 discloses a composition for the prevention and treatment of influenza virus, 2014-0141178 discloses an anti-influenza virus (hereinafter, referred to as " anti-influenza virus ") containing an extract of Lolium longanum as an active ingredient, Korean Patent Publication No. 10-2014-0115576 discloses an anti-influenza virus Korean Patent Laid-Open No. 10-2014-0143115 discloses a composition containing xylitol, ginseng or Hong There is disclosed a health food composition for preventing or ameliorating an influenza virus infection comprising any one or two or more selected from the group consisting of ginseng or red ginseng polysaccharide and ginseng or red ginseng saponin, Patent No. 10-1334143 discloses a composition for preventing or treating cold, avian influenza, swine influenza or swine flu, which contains polygala karensium extract and a detonated ternary compound separated therefrom, Japanese Patent Registration No. 10-0950445 discloses a compound which inhibits neuraminidase derived from licorice (Glycyrrhiza uralensis) and a composition for use in the prevention and treatment of avian influenza and swine flu comprising the same as an active ingredient Korean Patent No. 10-1177182 discloses that 95% horse mackerel extract And Korean Patent No. 10-1021830 discloses a swine influenza virus derived from an organic solvent extract of Clostacarix operculus and an antiviral agent against a new influenza virus Korean Patent No. 10-1317318 discloses an influenza inhibiting composition containing as an active ingredient an extract of Galla Rhois or a compound isolated therefrom, Korean Patent Laid-Open No. 10-2011-0132407 discloses an influenza- A plant extract composition for the prevention and treatment of influenza including an extract prepared from Radix isatidis and an extract prepared from Flos Lonicerae is disclosed in Korean Patent No. 10-0295395, , Antimicrobial agents for preventing and treating colds, including extracts of gingkohwa, peony, Korean Patent No. 10-0295389 discloses an antiviral agent containing extracts of quinces, undrawn citron ginger and horseradish, and Korean Patent Laid-Open No. 10-2003-0027133 discloses an antiviral agent containing black elderberry extract Korean Patent No. 10-1165592 discloses a medicament for preventing infectious rheumavirus infection which contains raspberry (Rubus idaeus), Fragaria vesca, blackberry (Rubus fruticosus), fig (Ficus carica) Chenopodium album, Agrimonia eupatoria, Eucalyptus globulus, Peach (Prunus persica), Apple (Malus pumila), Viola odorata, Lindera umbellata, Paullinia cupana Commelina communis, Capsella bursa-pastoris, Rabdosia japonica, Fragaria vesca, Marrubium vulgare, Marshmallow, Alfalfa spp. Aea officinalis, Plantago asiatica, Aloysia triphylla, Achillea millefolium, Cetraria islandica, Gynostemma pentaphyllum, and Petasites japonicus as active ingredients And Korean Patent No. 10-1086811 discloses a natural extract having an anti-influenza virus effect comprising one or two or more kinds of extracts selected from the extracts of Hibiscus, Ogapi, Korean Patent No. 10-1011454 discloses an influenza virus infection comprising an extract of Sophora flavescens, a fraction thereof, a terracarpane compound and a flavonoid compound or a pharmaceutically acceptable salt thereof isolated therefrom as an active ingredient A pharmaceutical composition for preventing and treating diseases is disclosed in Korean Patent Publication No. 10-2010-005265 No. 6 is an excellent antiviral active substance against the alga, Plenza virus A / H5N1. The methanol extract of the Asarum sieboldii root is suspended in distilled water, filtered and then separated with chloroform. Korean Patent No. 10-1203484 discloses a pharmaceutical composition for inhibiting avian influenza including Korean mistletoe. More particularly, the present invention relates to a pharmaceutical composition for inhibiting avian influenza including Korean mistletoe in a feed, 101115063 describes a composition for the prevention and treatment of influenza virus infection comprising an extract of Chrysanthemum morifolium, Korean Patent Laid-Open No. 10-2011-0041936 discloses a composition for preventing influenza virus infection comprising a rhusin-derived compound Korean Patent Laid-Open Publication No. 10-1087759 discloses a neurominidys bow containing a cedarwood extract And a pharmaceutical composition for the prevention and treatment of an influenza virus infection disease are disclosed in Korean Patent No. 10-1195120, and a composition for prevention and treatment of a highly pathogenic avian influenza H5N1 infection containing a lyophilized extract is disclosed in Korean Patent No. 10-1195120 Korean Patent No. 101189823 discloses a composition for the prevention and treatment of influenza virus infection comprising a polyphenolic compound, Korean Patent Publication No. 10-2011-0055852 discloses a composition for treating a virus containing an effective amount of flavonoid Korean Patent No. 10-1135946 discloses a composition for prevention and treatment of highly pathogenic avian influenza virus H5N1 virus infection containing a tandem extract, Korean Patent No. 10-1160743 discloses a composition for preventing or treating a highly pathogenic avian influenza H5N1 virus infection, Or a feed additive containing green tea as an active ingredient Korean Patent Laid-Open No. 10-2011-0040629 discloses a composition for preventing infection with a novel influenza A (H1N1) virus including a bank extract, and Korean Patent No. 101373219 discloses a composition for preventing infection of H1N1 virus A medicinal plant extract having an antiviral activity against influenza virus H1N1 as a fraction thereof after methanol extraction of a flower, passerine seedlings, and Hwanggi root-rooted cabbage plants has been described. In Korean Patent Publication No. 10-2012-0026851, Korean Patent Laid-Open No. 10-2011-0110381 discloses a pharmaceutical composition for preventing and treating influenza virus infectious diseases including plant extracts, which comprises a dry powder of a black triangle bud or its extract as an active ingredient, and a highly pathogenic avian influenza virus Have been disclosed, and a composition for preventing and treating Japanese Patent Application Laid-Open No. 10-1164515 discloses a method for producing a Chinese cabbage, a Chinese cabbage, a Chinese cabbage, a Chinese cabbage, a Chinese cabbage, a Chinese cabbage, a Chinese cabbage, a Chinese cabbage, a Chinese cabbage, a Chinese cabbage, , Korean Patent Laid-Open No. 10-2014-0030360 discloses a composition for preventing and improving influenza pseudo-symptoms and symptoms of infectious diseases comprising red ginseng as an active ingredient, Korean Patent No. 10-1387353 discloses a composition for the prevention and treatment of influenza virus-related diseases containing, as an active ingredient, an extract of Ivy leaf and a compound isolated therefrom, and Korean Patent No. 10-1369100 There is disclosed a composition for preventing and treating influenza virus-related diseases, which comprises an extract of a complex herb medicine consisting of ivy leaves and chrysanthemum as an active ingredient have.

Although the safety and efficacy of these anti-influenza viruses have been partially verified, the development of more effective anti-influenza virus agents is required for effective prevention and treatment of influenza viruses. Thus, the present inventors have found that when manuka honey, which is known to have skin moisturizing effect, skin soothing effect and antibacterial effect, is mixed with propolis and xylitol to extract a solvent, the antibacterial activity The present invention has been accomplished on the basis of the fact that it is remarkably large and particularly has an excellent effect for the prevention or treatment of symptoms of diseases caused by influenza virus.

The problem to be solved by the present invention is to develop a natural product combination agent which is effective for inhibiting and treating influenza viruses, not professional drugs.

The present invention provides an anti-influenza virus agent comprising, as an active ingredient, an extract extracted from a mixture of manuka honey, propolis and xylitol as the above-mentioned problem solution.

A preferred composition weight ratio of the mixture is 1 to 90% of manuka honey, 1 to 90% of propolis, and 1 to 90% of xylitol.

A preferred method for extracting the extract from the mixture is solvent extraction using water, 10-50% ethanol or 95% ethanol as a solvent.

When water is used as a solvent, the mixture is mixed with water at a weight ratio of mixture: water = 1: 10 to 30, and extracted with a pressure extractor at 100-120 ° C for 3-12 hours.

When 10 to 50% ethanol is used as a solvent, The mixture is mixed with 10-50% ethanol at a weight ratio of 10-50% ethanol = 1: 10-30, and extracted with a pressure extractor at 100-120 ° C for 3-12 hours. Ethanol is then evaporated to remove the components of ethanol and the remaining filtrate is used.

When 95% ethanol is used as a solvent, the mixture is mixed with 95% ethanol in a weight ratio of 1: 10-30: 95% ethanol and extracted for 30 to 90 days at room temperature.

According to the present invention, it has been confirmed through in vitro experiments and animal experiments that the deterioration of diseases due to colds can be prevented or treated. The anti-influenza virus agent according to the present invention can be used for influenza virus (H1N1) by examining the number of plaques after infecting an MDCK cell having a receptor for influenza virus on the cell surface for 1 hour at room temperature, In a plaque assay with inhibition test, it was confirmed that the inhibition of plaque formation by 97% ~ 66% depending on the concentration, as compared with the control (the group not reacting with the present invention), and it was confirmed that binding to influenza virus surface protein HA (Hemagglutinin) In the erythrocyte agglutination inhibition assay in which the present invention can inhibit the reaction between influenza virus and erythrocytes using erythrocytes capable of being used as target cells, it is known that when the present invention is applied at a high concentration, it inhibits the adhesion of influenza virus and erythrocytes Respectively. Further, as a result of the weight change rate test of the mouse, it was confirmed that the mice injected with the present invention started to gain weight faster than the mice not injected with the present invention after the virus infection, and were closer to the original body weight at a faster rate. Thus, it can be seen that the anti-influenza virus according to the present invention alleviates symptoms after influenza virus infection and has a shortening effect on the period of infection.

The present invention can be applied to a drink, a cold treatment tea, a rhinitis spray, a cold preventive soap, a cold preventive detergent and the like. In the case of natural product complexes, it is common to have effects on several similar viruses when they are effective against cold viruses, which will be revealed through continuous experiments. Accordingly, the anti-influenza virus agent according to the present invention can be applied to dogs for treating measles, feed mix for foot-and-mouth disease, avian influenza, and the like.

1 is a graph showing the relationship between the number of plaques generated when an influenza virus (N1N1) is mixed with an anti-influenza virus according to the present invention and MDCK cells, This chart shows the incidence rate.
FIG. 2 is a view showing whether erythrocyte sedimentation occurs when influenza virus reacts with red blood cells or inhibits the reaction in a hemagglutination inhibition assay. FIG.
FIG. 3 is a hemagglutination inhibition assay result obtained by analyzing erythrocyte sedimentation state by mixing with influenza virus (N1N1) and reacting with anti-influenza virus (anti-influenza virus) according to the present invention.
Fig. 4 is a chart of percent change in weight of the influenza virus-infected group of Tamiflu, the group administered with the present invention and the group administered with D / W.

The present invention provides an anti-influenza virus agent comprising as an active ingredient an extract extracted from a mixture of manuka honey, propolis and xylitol.

Honey has not only been used as food for thousands of years, but has also been used for medical purposes, primarily as an antibacterial agent. The main cause of the antibacterial active substance is the hydrogen peroxide produced in the honey by the enzyme glucose oxidizing agent. Although honey has a clear antibacterial effect, the antibacterial efficacy of honey varies substantially depending on the type of honey. Most of the compounds present in honey are included in the group of carbohydrates, enzymes, aromatic acids, hydrocarbons, straight-chain monovalent and divalent base acids and water. Honey is mainly composed of sugar glucose, fructose, sucrose and maltose, and contains many other oligosaccharides. The other substance added to the honey by bees is the amino acid with the most abundant proline and a small amount of catalase. These enzymatic catalase break down hydrogen peroxide into water and oxygen. Diacids including octandedioic, nonanedioic, decanedioic, and trans-2-decenedioic have also been reported to be present in honey.

The Manuka tree, Leptospermum scoparium, is native to New Zealand and can grow to a height of 6-8 m and a diameter of 7-10 cm, These flowers produce honey with a very strong aroma, called manuka honey. Manuka Honey not only moisturizes dry skin, it also has better antimicrobial action against damaged skin and Helicobacter pneumonia.

Manuka honey has an additional antibacterial active substance in addition to the antibacterial active substance of common honey. These additional active substances are known as peroxide active materials and are referred to as Unique Manuka Factors (UMF). These non-peroxide active substances, which contain large amounts of manuka honey, have a higher level of antibacterial activity that can not be accounted for by osmolarity, pH and glucose oxidant activity alone. There have been many studies to characterize the non-peroxide active substances (UMF) of manuka honey, and attempts have been made to identify the causative segments of these additional active substances, but despite many years of research, Non-peroxide components were not isolated or identified. In recent years, hydrogen peroxide is the only significant antibacterial substance of honey, hydrogen peroxide is produced by glucose oxidizing agents in honey or their segments, and other substances such as propolis-derived phenolic systems are not important compared to hydrogen peroxide, Studies have shown that the level of hydrogen peroxide is essentially determined by the amount of plant-derived catalase in honey, and Manuka honey has more plant-induced catalase than other honey. However, these findings also do not provide sufficient explanation or evidence for the presence or absence of the specific Manuka Factor (UMF).

However, many studies have shown that such Manuka Honey Additional Active Substances (UMF) can be stable to heat and light, soluble in organic solvents such as ethanol and ether, The degree of activation was confirmed. The present invention utilizes these properties revealed for manuka honey to reveal an immune enhancement effect that enhances a direct antiviral effect or an antiviral effect, thereby extracting a new natural product complex effective for inhibiting and treating influenza virus.

The present invention uses propolis, an antibacterial agent extracted from a beehive, and xylitol, a pentose, to elicit an anti-influenza virus effect from Manuka honey.

Propolis is used as an antibacterial agent in beehives as a resinous material collected by bees from the gum exudate of trees. So far, it is known that Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Trichomonas bacteria, Salmonella and the like are effective. The honey bee protects itself against germs and viruses by spraying propolis in the cracks of the honeycomb, and prevents the invasion of enemies such as wasps or rats. Especially when the queen bee spawning, worker bees are used to clean the spawning place. Propolis includes substituted benzoic acid, cinnamic acid, and flavonoids. Flavonoids are the major antibacterial agents in propolis. Flavonoids identified as causative agents for antibacterial active substances include galangin, pinocembrin, caffeic acid and ferulic acid. The main efficacy of propolis is antibacterial, anti-inflammatory, analgesic, immune-enhancing, anti-cancer, and antioxidant. The anti-inflammatory effect is because it reduces the enzymes that cause prostaglandins, which cause inflammation in people's bodies, by up to half. The main ingredient, flavonoid, has antioxidant effect because it eliminates active oxygen. And quercetin, which has an anti-cancer effect, and blocks cancer genes before their genes are replicated.

The present invention was added to the mixture in anticipation of having an antiviral effect directly on the propolis or an immunostimulating effect enhancing the antiviral effect.

Xylitol is extracted from the bark and sap of five decadic species (Betula platyphylla var. Japonica Hara, Birch), Betula platyphylla Sukatschev (Manchuria birch), etc., belonging to the Betulaceae family (Scientific name: Betula platyphylla var. Japonica Hara) (C ₅ H ₁₂ O _5, molecular weight 152.15). The present invention uses xylitol to increase sugar density of the extract.

In Oriental medicine, birch has been known to have beneficial effects on skin and sap. It is known that birch bark is called as white bloom, bark, flower bark, etc. and has effects such as fever, water, detoxification, and extermination. Bronchitis, pneumonia and hepatitis, jaundice, nephritis, cystitis and so on. It is effective for bronchitis, pneumonia and hepatitis caused by the cold. Most of the cases of cold or hepatitis are viral diseases. Therefore, the birch bark or sap extract has a direct antiviral effect or an immune enhancing effect . Xylitol is a pentose (C ₅ H ₁₂ O ₅ ) extracted from the bark or sap of birch, and is expected to maximize the effect of the Manuka honey virus when included in the mixture. Xylitol is a pentose (C ₅ H ₁₂ O ₅ ) rather than a hexose (C ₆ H ₁₂ O ₆ ) that bacteria prefer, and the antiviral properties of Manuka honey are more strongly expressed as the sugar density is higher.

The above-mentioned substances of Manuka Honey, Propolis and Xylitol have long been used for the treatment of infectious diseases caused by pathogenic microorganisms including bacteria. Most antimicrobial and immune enhancing effects have been demonstrated, and it is anticipated that there will be direct anti-influenza virus effects or anti-influenza virus enhancing effects, Respectively.

&Lt;

<In vitro experiments>

Whether or not the anti-influenza virus according to the present invention interferes with the infection of influenza virus (H1N1) and whether or not influenza virus inhibits adherence to target cells is determined using MDCK cells Plaque assay, hemagglutination assay, and hemagglutination assay.

1. Preparation of sample

A mixture of 100 g of Manuka honey, 10 g of propolis and 600 g of xylitol was prepared, and the mixture was mixed with 50% ethanol in a weight ratio of mixture: 50% ethanol = 1: 20 and extracted with a pressure extractor at 120 ° C for 8 hours . Ethanol was evaporated to remove the components of ethanol, and the remaining filtrate was obtained as a sample. The sample thus obtained was centrifuged at 14,000 rpm / 10 min / 4 ° C, filtered through 0.22 μm filtration, and stored at 4 ° C.

2. Preparation of influenza virus

Human influenza virus A / PR / 8/34 (H1N1) was harvested after inoculation at 10 days of fertilization and incubation at 37 ° C for 48 hours.

3. MDCK cell preparation

MDCK cells were cultured by adding 10% heat-inactivated FBS to MEM media.

3. Plaque Assay ( Sputum examination )

In order to examine how the influenza virus reacted with the anti-influenza virus of the present invention affects cell infections and proliferation, about 100 PFU / ml of influenza virus and the anti-influenza virus of the present invention were administered at various concentrations (1/10, 20, 1/40, 1/80, 1/160, 1/320, 1/640, 1/1280, 1/2560, 1/5120, 1/10240) at room temperature for 1 hour. After the reaction, the influenza virus was adsorbed to the MDCK cells at room temperature for 45 minutes (330 μl / well). After 45 minutes, the virus was removed and overlay medium (DMEM + 1% agarose + 10 μg / ml trypsin) was dispensed. Plates were incubated at 37 ° C for 2-3 days and then stained with 1% crystal violet to determine the number of plaques formed. The control (control) was obtained by adsorbing influenza viruses not influenced by the anti-influenza virus of the present invention on MDCK cells. The virus inhibition efficacy of each drug was determined by comparing the number of plaques generated in the plaque and the number of plaques generated in the experimental group.

FIG. 1 shows the number of plaques generated when MDCK cells were mixed with an influenza virus (N1N1) while varying the concentration of the anti-influenza virus according to the present invention, compared with the number of plaques generated when only influenza virus was administered , And a plaque occurrence ratio. As shown in Figure 1, the experimental group (1/10, 1/20, 1/40, 1 / 40th in the x-axis) was significantly lower than the control group (leftmost bar marked with 0 in the x- 80, 1/160, 1/320, 1/640, 1/1280, 1/2560, 1/5120, 1/10240) showed that the number of plaques was reduced by 50% or more at a high concentration (1/10 to 1/80) I could. Therefore, the anti-influenza virus agent according to the present invention has a very effective inhibitory effect on influenza virus infection.

4. Hemagglutination Inhibition Assay (Red blood cell coagulation disturbance analysis)

As shown in FIG. 2, generally, influenza virus is bound to red blood cells by hemagglutinin, which is a surface protein, and erythrocyte sedimentation does not occur. However, when influenza virus is not bound to erythrocytes, erythrocytes aggregate and precipitate.

To ensure that the anti-influenza virus agent of the present invention can inhibit the adhesion of a influenza virus in a target cell, 2-fold serial dilution (Dilution factor for anti-influenza viral agent of the present invention to 25㎕ PBS: ² ^1, 2 2 , 2 ³ , 2 ⁴ , 2 ⁵ , 2 ⁶ , 2 ⁷ ) and 4 HAU (Hemagglutination unit) influenza viruses were mixed in the same amount and reacted at 37 ° C. for 1 hour. Then, 1% chick RBC (Red Blood Cell) was dispensed at 50 μl / well and allowed to stand at room temperature for 30 minutes. RBC precipitation was observed.

3 is 1 is diluted to an anti-influenza viral agent according to the invention the concentration drain 2 ^1, 2 ^2, 2 ^3, 2 ^4, 2 ^5, 2 ^6, is mixed with the influenza virus (N1N1) and otherwise to ²⁷ red blood cells (Hemagglutination Inhibition Assay). The results of the hemagglutination inhibition assay are shown in FIG. Influenza virus surface protein HA (Hemagglutinin) induces binding with target cells, which leads to the onset of infection and has a great influence on disease susceptibility. As a result of examining whether each of the anti-influenza viruses of the present invention can inhibit the reaction between influenza virus and erythrocytes using erythrocytes capable of binding to HA as target cells, the present invention was applied at an appropriate concentration (2 ^{5 in the} experimental example) , It was confirmed that the influenza virus interferes with the adhesion of red blood cells. This means that the virus can directly interfere with the attachment of the virus to the target cell.

&Lt; Animal test -

In vitro experiments of the anti-influenza virus of the present invention presumed that the influenza virus had a clinical effect and the experiment was conducted with an animal model to obtain scientific data on the influenza virus A (H1N1) of the present invention anti-influenza virus .

1. Preparation of sample

The sample from the intracellular experiment was concentrated 10 times and stored as a storage sample (10X stock). The sample was stored 3 times and used again.

2. Preparation of influenza virus

Human influenza virus A / PR / 8/34 (H1N1) was inoculated into fertilized eggs at 10 days of age and incubated at 37 ° C for 48 hours to harvest the influenza virus.

3. Preparation of mouse

Balb / c 6-week-old female mice were used as animal models. The experiment was initiated by administering the sample over an adaptation period of about 2 days.

4. Investigation of the rate of change in weight of mice

Mice were infected with influenza virus at a concentration of 0.2 LD ₅₀ , and the weight change of each group was examined.

Five mice per group were administered with the anti-influenza virus of the present invention in the experimental group, Tamiflu in the positive control group (Tamiflu administration group), and distilled water (D / W) in the Negatve control group did. In the experimental group, the anti-influenza virus 10X stock of the present invention as a sample was concentrated 3-fold and administered at 100 μl / once. In the positive control group (Tamiflu group), Tamiflu was administered at 10 mg / kg / day. In the negative control group (control), 100 μl of sample D / W was administered per synapse. The administration was orally administered twice a day, once in the morning and in the afternoon, from virus challenge -1 Day.

The virus challenge was an intranasal challenge (nasal infection) with 0.2LD ₅₀ of influenza virus after intravenous injection of zoletil (anesthetic agent) into mice. Tamiflu was administered until 8 days after virus challenge, and the distilled water (D / W) and the samples of the present invention continued to be administered until the end of the experiment.

Weighing was done once every 9 ~ 10 o'clock in the morning and after 14 days from virus challenge, the experiment was finished. The body weight was measured daily and the body weight change rate was measured based on the weight before the virus infection.

FIG. 4 is a graph showing the effect of the anti-influenza virus (D / W), anti-influenza virus, and anti-influenza virus of the present invention on the susceptibility to influenza virus A / PR / 8 / The percent change in body weight profile of influenza virus effect was investigated. Values represent mean ± SEM of 5 per group.

As shown in FIG. 4, in the Tamiflu group after influenza virus infection, the body weight of the experimental group and the control group decreased from 5 days after challege, and recovery was observed from the experimental group after 10 days. The experimental group receiving the anti - influenza virus of the present invention had significantly less weight loss rate and faster recovery time than the control group.

Table 1 below is a numerical table comparing percent body weight change from day 9 after the virus challenge in which the difference in body weight between the experimental group infected with influenza virus A / PR / 8/34 (H1N1) and the control group was remarkably observed. The values mean mean SEM.

Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Control group 77.28 1.55 73.94 + 1.97 75.49 + - 3.87 78.06 + - 5.30 81.97 + - 7.51 85.33 + - 8.02 Experimental group 85.12 ± 1.16 85.67 ± 3.21 88.51 + - 4.71 91.50 ± 4.68 91.66 ± 3.69 97.73 ± 3.33

As a result, when the anti-influenza virus of the present invention was administered, it was found that it became closer to the Tamiflu group on the 14th day. Thus, the ingestion of the anti-influenza virus of the present invention showed a significant effect of alleviating symptoms and shortening the period of infection after influenza virus infection.

Claims

An anti-influenza virus agent comprising, as an active ingredient, an extract derived from a mixture of manuka honey, propolis and xylitol.