KR102110503B1 - Process for Compositions of Disinfection for Low Pathogenic Avian Influenza Virus Including Extract of Pleurotus nebrodensis - Google Patents

Abstract

The present invention is 1) a first step of obtaining a giant mushroom extract by cutting into a suitable size, extracting using ethanol as a solvent, filtering and freeze-drying; 2) a second step of cutting the perilla leaf to an appropriate size, extracting using ethanol as a solvent, filtering and freeze-drying to obtain a perilla leaf extract; 3) a third step of sintering shellfish, crushing, meshing, and then ultrasonically pulverizing to nano-particle, and then electrolyzing to ionize the mineral particles; 4) Purified water, the royal mushroom extract obtained in the first step, the perilla leaf extract obtained in the second step, sodium cyanate (NaOCN), glutaraldehyde (C ₅ H ₈ O ₂ ) in the ionizing mineral nanoparticles in the third step, A fourth step of making a mixed solution by mixing guar hydroxypropyltrimonium chloride and citric acid (C ₆ H ₈ O ₇ ); 5) a fifth step of nano-splitting the mixed solution made in the fourth step so as not to be precipitated by ultrasonic pulverization; relates to a method for producing a low-pathogenic avian flu virus disinfectant composition.
In addition, the present invention is purified water 95.5 parts by weight of the king mushroom extract 5.0 ~ 7.0 parts by weight, perilla leaf extract 3 parts by weight, mineral particles 2 parts by weight of sodium cyanide (NaOCN) 1 part by weight, glutaraldehyde (C ₅ H ₈ O ₂ ) 0.5 parts by weight of Guar Hydroxypropyltrimonium Chloride, 0.5 parts by weight of citric acid (C ₆ H ₈ O ₇ ) It relates to a low pathogenic bird flu virus disinfectant composition.
The low-pathogenic bird flu virus disinfectant composition containing the large mushroom extract, perilla leaf extract and ionized mineral nanoparticles according to the present invention has the advantage of not only having excellent disinfecting effect against the low-pathogenic bird flu virus but also having little effect on environmental pollution. .

Description

Process for Compositions of Disinfection for Low Pathogenic Avian Influenza Virus Including Extract of Pleurotus nebrodensis}

The present invention relates to a method for preparing a low pathogenic avian influenza virus disinfectant composition containing a giant mushroom extract and a low pathogenic avian influenza virus disinfectant composition prepared therefrom. More specifically, a method for producing a low pathogenic avian influenza virus disinfectant composition comprising low-pathogenic bird flu extracts, perilla extract and mineral particles ionized by nano- and electrolysis by ultrasonic pulverization and low-pathogenic avian flu prepared therefrom The present invention relates to a virus disinfectant composition.

The avian influenza virus is a virus that infects poultry (chicken, turkey, duck, etc.), and there is also a human influenza virus, a swine influenza virus, a horse influenza virus, and mainly corresponds to an envelope virus. Depending on the degree of pathogenicity of the virus, it can be largely divided into low pathogenicity and high pathogenic avian influenza. In addition, the type of influenza virus is classified into type A, type B, and type C according to the components (matrix, nucleoprotein) of the virus. Type A and B are for humans and type A is for animals other than humans. Cause disease.

The serotypes of type A influenza are classified according to two types of proteins (Hemagglutinin, Neuraminidase), and there are H and N serum types. The serotypes of influenza virus are indicated by respective H and N serotypes, for example, H9N2. Influenza virus has 16 subtypes from H1 to H16, and N serotype is divided into 9 subtypes from N1 to N9, and influenza virus that can be arithmetic when H type and N type are combined There are 144 serotypes (16 × 9). Serotypes are represented by H3N2, H9N2, etc., and have a significant association with the pathogenicity of the virus.

Each serotype does not have a cross-immune response or is weak so that it cannot prevent other serotypes from influenza virus infection, that is, chickens immunized with the H6N1 serotype virus cannot prevent infection of other serotypes such as H5N2. Vaccines are difficult to develop because influenza viruses with various serotypes do not have defense-related antigens regardless of serotype. In addition, bird flu occurs repeatedly every year, not only in Korea, but around the world, but it is difficult to develop timely disinfectants. Therefore, prior to proactively, H6N1, H5N1, H9N2, H5N6, etc. Various combinations of individual antiviral efficacy evaluations and compositions for each serotype are urgently needed worldwide.

As a related art, the following patent document 0001 discloses “a sterilizing and killing virus composition that improves disinfecting power, including citric acid and tartaric acid in hepatitis and malic acid,” but it is disclosed that avian influenza virus is a type of virus. It is not specific. In addition, since the composition is made of natural materials and biodegradable by microorganisms is possible, it has little effect on environmental pollution, and has a disadvantage in that the disinfecting effect against the low pathogenic bird flu virus is not excellent.

In addition, Patent Document 0002 below discloses a disinfecting method of inactivating avian influenza virus by contacting a drug with an active ingredient of hoon liquor obtained from wood to an object to be sterilized, but is limited to specific bacteria. It is not. This has the disadvantages that the composition is made from natural materials, so that the load on the global environment is very small, there is no concern for the safety of humans and animals, and the disinfecting effect against the low pathogenic bird flu virus is not excellent.

In addition, Patent Document 0003 below discloses “a composition for disinfecting algae influenza including sodium metasilicate and potassium carbonate”, but this is only a very general disinfection composition for disinfecting algae housing and various livestock equipment and facilities. It is not a composition for disinfecting avian influenza virus that is not specific to a low pathogenicity specific serotype.

Accordingly, the present inventors, while studying disinfectants related to the low pathogenic avian influenza virus unlike the prior art, mixed the nanoionized by ultrasonic grinding and ionized minerals by electrolysis as an active ingredient during the study of disinfectants related to the low pathogenic avian influenza virus to specific serotypes of low pathogenicity. It was found that the disinfecting effect is excellent, and the present invention was completed.

Korea Patent Publication No. 10-2009-0009442 (2009. 01. 13.) Japanese Patent Application Publication No. 2013-136610 (2012. 01. 17.) Korean Registered Patent Publication No. 10-0772054 (October 25, 2007)

An object of the present invention is to provide a method for preparing a low pathogenic avian influenza virus disinfectant composition containing a giant mushroom extract.

The present invention is 1) a first step of obtaining a giant mushroom extract by cutting into a suitable size, extracting using ethanol as a solvent, filtering and freeze-drying; 2) a second step of cutting the perilla leaf to an appropriate size, extracting using ethanol as a solvent, filtering and freeze-drying to obtain a perilla leaf extract; 3) a third step of sintering shellfish, crushing, meshing, and then ultrasonically pulverizing to nano-particle, and then electrolyzing to ionize the mineral particles; 4) Purified water, the royal mushroom extract obtained in the first step, the perilla leaf extract obtained in the second step, sodium cyanate (NaOCN), glutaraldehyde (C ₅ H ₈ O ₂ ) in the ionizing mineral nanoparticles in the third step, A fourth step of making a mixed solution by mixing guar hydroxypropyltrimonium chloride and citric acid (C ₆ H ₈ O ₇ ); 5) a fifth step of nanoparticles so as not to be precipitated by ultrasonic grinding the mixed solution prepared in the fourth step; provides a method for producing a low pathogenic bird flu virus disinfectant composition comprising a.

In addition, the present invention is purified water 95.5 parts by weight of the king mushroom extract 5.0 to 7.0 parts by weight, perilla leaf extract 3.0 parts by weight, ionized mineral nanoparticles 2.0 parts by weight, sodium cyanide (NaOCN) 1.0 parts by weight, glutaraldehyde (C ₅ H ₈ O ₂ ) 0.5 parts by weight, Guar Hydroxypropyltrimonium Chloride (Guar Hydroxypropyltrimonium Chloride) 0.5 parts by weight, citric acid (C ₆ H ₈ O ₇ ) It provides a low pathogenic bird flu virus disinfectant composition comprising 0.5 parts by weight.

Other specific examples for implementing the present invention are included in the detailed description below.

The method of manufacturing a low pathogenic avian flu virus disinfectant composition containing the extract of the king mushroom according to the present invention is a low pathogenic avian influenza virus disinfectant composition containing ionized by electrolysis and nano-processing of various minerals obtained from the king mushroom extract, perilla leaf extract and shellfish As a method for manufacturing a, it has an advantage of not only having a very good disinfecting effect against the low pathogenic avian flu virus but also having little effect on environmental pollution.

Hereinafter, a preferred embodiment and a property measurement method will be described in detail with respect to a method for preparing a low pathogenic avian influenza virus disinfectant composition containing a giant mushroom extract of the present invention. The present invention can be better understood by the following examples, and the following examples are intended to explain in detail that a person skilled in the art to which the present invention pertains can easily implement the invention. Therefore, it does not mean that the technical spirit and scope of the present invention are limited.

In the present invention, "disinfection" means killing pathogens to prevent infection or transmission of disease In addition, in the present invention, the "microorganism" is included as an active ingredient in the low-pathogenic avian flu virus disinfectant composition of the present invention This is a concept that includes all the bacteria, fungi, yeast, and algae that have a disinfecting effect.

Hereinafter, a method for preparing a low pathogenic avian influenza virus disinfectant composition containing the giant mushroom extract of the present invention will be described in more detail.

Method for preparing a low pathogenic avian influenza virus disinfectant composition containing a blue mushroom extract according to an embodiment of the present invention is 1) extract the blue mushroom using ethanol as a solvent, filter and freeze-dried to obtain a blue mushroom extract Step 1; 2) a second step of cutting the perilla leaf to an appropriate size, extracting using ethanol as a solvent, filtering and freeze-drying to obtain a perilla leaf extract; 3) a third step of sintering the shellfish, crushing and meshing, and then ultrasonically pulverizing the nanoparticles, followed by electrolysis to ionize the mineral nanoparticles; 4) Purified water, the royal mushroom extract obtained in the first step, the perilla leaf extract obtained in the second step, sodium cyanate (NaOCN), glutaraldehyde (C ₅ H ₈ O ₂ ) in the ionizing mineral nanoparticles in the third step, A fourth step of making a mixed solution by mixing guar hydroxypropyltrimonium chloride and citric acid (C ₆ H ₈ O ₇ ); 5) a fifth step of nano-milling the mixed solution made in the third step so as not to be precipitated by ultrasonic pulverization.

The first step is a preparation step in the method for preparing a low pathogenic avian influenza virus disinfectant composition of the present invention, cutting the toadstool to an appropriate size, extracting using ethanol as a solvent, filtering and freeze-drying to extract the toadstool extract. It is a step to get.

The second step is a step of cutting a perilla leaf into an appropriate size, extracting using ethanol as a solvent, filtering and freeze-drying to obtain a perilla leaf extract.

The giant mushroom (Pleurotus nebrodensis) used in the present invention is a type of sub-mushroom called the sub-mushroom of the genus Oyster mushroom, and is called the name of the giant mushroom by utilizing genetic properties different from those of the conventional sub-mushroom. Among the edible mushrooms, it is known to have the largest size with a price of 2-3 cm and a diameter of 10-15 cm, and the nutritional content is 10-40 times higher than that of similar mushrooms. In particular, Daewang Mushroom is a natural food with medicinal value because it contains a large amount of amino acids, vitamins, and minerals, and is known to remove harmful oxygen that causes degenerative diseases such as dementia, stroke, rheumatism, and diabetes.

The term "perilla leaf" refers to the leaves of a perennial herb belonging to the genus Lamiaceae, and the perilla leaf can use any perilla leaf commercially available in Korea, preferably domestic perilla leaves, without limitation.

The large mushroom extract or perilla leaf extract of the present invention can be extracted by not destroying the active ingredient or at room temperature or by heating in a minimized condition. The king mushroom or perilla leaf extract may be obtained by refluxing the king mushroom or perilla leaf with 70Vol% ethanol, respectively, at 80 ° C for 50 minutes and 30 minutes.

The large mushroom extract, perilla leaf extract, respectively, is preferably extracted by adding an extraction solvent of 2 to 20 times, preferably 5 to 15 times the dry weight of the giant mushroom and perilla leaf, most preferably 10 times the weight. . Water is generally used as the extraction solvent, but other organic solvents other than water may be used as necessary. Examples of the organic solvent that can be used include C ₁ to C ₄ lower alcohols, but are not limited thereto. Preferably, ethanol and its aqueous solution are mentioned, and most preferably, it is 70Vol% of ethanol. In the present invention, the "Daily mushroom extract", "perilla leaf extract" is preferably a giant mushroom extract and perilla leaf extract prepared using the extractant, respectively, the amount of their addition in the disinfectant composition of the present invention is 5.0 ~ It is preferable to use 7.0 parts by weight and 3.0 parts by weight. At this time, when adding less than 5.0 parts by weight of the mushroom extract, algal flu virus suppressing effect is somewhat insufficient, and adding more than 7.0 parts by weight has an excellent virus suppressing effect, but has a disadvantage of increasing the manufacturing cost of the composition.

In the present invention, the "Daily mushroom extract" or "Perilla leaf extract" is preferably a giant mushroom extract or perilla leaf extract prepared using the extraction solvent. The blue mushroom extract or perilla leaf extract extracted using ethanol solvent can be filtered to improve the quality to remove debris, etc., to prevent deterioration, and freeze-dried for preservation.

The third step is a step of sintering shellfish, crushing, meshing, and then ultrasonically pulverizing to nano-particle, and then electrolyzing to ionize the mineral particles.

First, the shellfish is not limited because any kind of shellfish that can be obtained from the beach can be used, but among them, it is preferable to use a scallop or cockleshell. The calcined mineral obtained by firing the shellfish at 1,200 ° C. for 2 hours can be pulverized into a pulverizer to form a powder, and contains Mg, Na, K, P, S, Fe, and Zn as its components. The particle size is preferably a powder of 30 to 300 mesh, and when the particle size is less than 30 mesh, the price is expensive, so there is a problem in economic efficiency, and when it exceeds 300 mesh, there is a problem of precipitation without melting.

Then, ultrasonic pulverization (sonication) is performed first by repeatedly applying 25 W of ultrasonic waves to the meshed mineral particles three times for 10 minutes. Through the ultrasonic pulverization process, nanoparticles having a size of 100 nm could be obtained, and the particle size did not change even if it was kept at room temperature for 7 days in this state (see the experimental results [Table 1]). After the nanonization process through the ultrasonic pulverization process, the temperature was slightly increased while stirring at 100 rpm while the mineral particles were dissolved in water to make the condition 45 to 50 ° C., followed by the (+) and (−) poles. Electrolysis of 0.1 ~ 5V through electrolytic rod is induced for 3 ~ 5 times in total for several minutes. Electrolysis of water in the state where the mineral particles are dissolved promotes ionization of the minerals, thereby inhibiting the precipitation of each of the mineral particles and maintaining the ionized state. At this time, the following reaction occurs at the cathode and the anode, and ionization of the minerals proceeds in this process.

(1) cathode _{(Cathode): 2H 2 O +} 2e - -> 2OH - + H 2

(2) The positive electrode ^{(Anode): 2OH - ->} H 2 O + 2e - + 1 / 2O 2

On the other hand, the nanonization and electrolysis performed above raise the solubility of water and minerals, and cause an effect of maintaining solubility even after a considerable time (about 4 weeks or more) after dissolution. As a result of experiments over a long period of time after separate nanoparticles and electrolysis of the aqueous solution of mineral particles, nanoparticle size changes were observed within an error range of less than 10% by maintaining 105 to 110 nm even after 270 days at room temperature. It can be seen that it is maintained in a relatively stable state (see Table 1 for experimental results). In addition, the addition amount of the disinfectant composition of the present invention is preferably 2.0 parts by weight.

 Particle size by storage period of mineral particles Storage temperature Storage period (days) Particle size (nm) Room temperature 25 ± 2 ℃ 0 100 7 101 ~ 103 Room temperature 30 100 60 101 90 102 180 104 240 108 270 110

In the fourth step, purified water, a king mushroom extract, a perilla leaf extract obtained in the first and second steps, sodium cyanate (NaOCN) and glutaraldehyde (C ₅ H ₈ O ₂ ) in the ionizing mineral nanoparticles in the third step , Guar Hydroxypropyltrimonium Chloride, Citric acid (C ₆ H ₈ O ₇ ) is mixed to make a mixed solution. At this time, 95.5 parts by weight of purified water, 5.0-7.0 parts by weight of the king mushroom extract, 3.0 parts by weight of the perilla leaf extract, 2.0 parts by weight of mineral particles, 1.0 parts by weight of sodium cyanate (NaOCN), glutaraldehyde (C ₅ H ₈ O ₂ ) 0.5 parts by weight Part, a mixed solution can be prepared by containing 0.5 parts by weight of Guar Hydroxypropyltrimonium Chloride and 0.5 parts by weight of citric acid (C ₆ H ₈ O ₇ ).

The sodium cyanide (NaOCN) is a white crystalline solid, and in the disinfectant composition of the present invention, is a substance used as an oxidizing agent that destroys proteins, such as viruses, by oxidation. In addition, the addition amount of the disinfectant composition of the present invention is preferably 1.0 part by weight.

Since the glutaraldehyde (C ₅ H ₈ O ₂ ) is toxic, it should not be directly contacted with humans and livestock, and its addition amount is preferably used in 0.5 parts by weight. In addition, it has a good disinfection effect even if there are some organic matters, but it has a disadvantage in that it is expensive, so it is expensive to use in large quantities. This means that the disinfecting effect is maintained even if not added.

The citric acid is contained as a free acid in the seeds or juices of many plants, and is widely used for various purposes, such as fruit aroma supplements, preservative enhancers, and pH adjusters. It is safe for people and clothes, so it has a good effect, but its penetration is weak, so the effect is very low in the presence of organic matter. The addition amount is preferably used in 0.5 parts by weight, which means that the disinfecting effect is maintained even without addition.

The fifth step is a step of nano-sizing the mixed solution made in the fourth step so as not to be precipitated by ultrasonic grinding. It is preferable to perform the ultrasonic pulverization (sonication) of adding 5 W of ultrasonic waves to the mixed solution made in the fourth step in 3 repetitions for 10 minutes to nano-scale to help solution of the mixture and to prevent precipitation from occurring.

The first to fifth steps were sequentially performed to produce 95.5 parts by weight of purified water, 5.0-7.0 parts by weight of the king mushroom extract, 3.0 parts by weight of the perilla leaf extract, 2.0 parts by weight of ionized mineral nanoparticles, and 1.0 parts by weight of sodium cyanide (NaOCN). , 0.5 parts by weight of glutaraldehyde (C ₅ H ₈ O ₂ ), 0.5 parts by weight of Guar Hydroxypropyltrimonium Chloride, 0.5 parts by weight of citric acid (C ₆ H ₈ O ₇ ) to prevent precipitation A low pathogenic avian flu virus disinfectant composition can be prepared.

On the other hand, the low pathogenic avian flu virus disinfectant composition containing the extract of the king mushroom of the present invention, as long as it does not inhibit the disinfecting effect of the active ingredient, the dispersing agent, carrier and other additives such as other antimicrobial agents It may be further included.

For example, in the case of dispersants, water, alcohol (e.g. methyl alcohol, ethyl alcohol, ethylene glycol, propylene glycol, diethylene glycol, glycerin, etc.), ketones (e.g. acetone, methyl ethyl ketone, etc.), ethers (e.g. Dioxane, tetrahydrofuran, cellosolve, diethylene glycol dimethyl ether, etc., aliphatic hydrocarbons (e.g. hexane, kerosene, etc.), aromatic hydrocarbons (e.g. benzene, toluene, xylene, naphthalene, methyl naphthalene, etc.), halogenated hydrocarbons (E.g. chloroform, carbon tetrachloride, etc.), acid amides (e.g. dimethyl formadid, etc.), esters (e.g. methyl acetate esters, ethyl acetate esters, butyl acetate esters, fatty acid glycerin esters, etc.), nitrols (e.g. Acetonitrile, etc.), surfactants (higher alcohol sulfate esters, alkyl sulfonic acids, alkyl allyl sulfonic acids, quaternary ammonium salts, oxy alkylamines, fatty acids Thermal switch, a polyalkylene oxide compound, anhydro-sorbitol compounds), but can be one or two or more selected from, but is not limited thereto.

In the case of carriers, clay (e.g., kaolin, bentonite, acid clay, etc.), talc (e.g., talc powder, pyrite powder, etc.), silica (e.g., diatomaceous earth, silicic anhydride, mica powder, etc.), alumina, sulfur powder and activated carbon Among them, one or two or more may be selected, but is not limited thereto.

The low pathogenic avian influenza virus disinfectant composition containing the giant mushroom extract of the present invention may be prepared in a liquid or solid phase, and the disinfectant composition of the present invention may be administered in any manner, such as oral or parenteral. Oral dosage forms include tablets, pills, powders, liquids, and food forms. Parenteral dosage forms are injections, topical dosages (creams, ointments, etc.), suppositories, and sprays (when applied to plants). And the like.

The disinfectant composition of the present invention is preferably administered by topical administration, and is applied to H9N2, a low-pathogenic avian influenza virus, and may be administered / sprayed to and around birds and bird colonies and breeding grounds.

In addition, a tissue made of a mixed material of pulp and non-woven fabric having a size of 20 cm × 10 cm in width and length is placed in the disinfectant composition according to the present invention and soaked for 1 to 2 hours so that the disinfectant composition is sufficiently absorbed in the tissue. An antiviral cleaning tissue impregnated with the disinfectant composition according to the invention may be prepared. When the face and hands are wiped using the cleaning tissue prepared as described above, it has been shown that the anti-viral effect is greatly improved compared to the case where the exposed skin is washed with a hand sanitizer.赤) It was confirmed through skin irritation test that no skin damage shape was observed.

Hereinafter, the present invention can be better understood by the following manufacturing examples and examples, and the following manufacturing examples and examples are for illustrative purposes of the present invention and are not intended to limit the protection scope of the present invention.

<Production Example>

  1. Experimental materials

    In order to verify the antiviral effect of the test substance against the avian influenza virus (AI virus), the experimental animal used 2-3 weeks old SPF (specific pathogen free) chicken, SPF-based fetal egg, and the used virus was low pathogenic algae influenza (LPAI). -H9N2) was used.

  2. Preparation of Daewang mushroom extract

     The giant mushrooms were purchased from each drugstore. After cutting the giant mushrooms to an appropriate size by 1000 g, 10L of 70Vol% ethanol was used as a solvent and extracted for 50 hours at 80 ° C with a reflux extractor. The extract was filtered with Whatman filter paper, and 31.3 g of each extract of dry weight was obtained using a lyophilizer.

  3. Preparation of perilla leaf extract

     Perilla leaves were purchased at a large supermarket. After cutting 1000 g of perilla leaves into an appropriate size, 10 L of 70 Vol% ethanol was used as a solvent and extracted at 80 ° C. for 30 minutes using a reflux extractor. The extract was filtered with Whatman filter paper, and 32.2 g of dry weight extract was obtained using a lyophilizer.

  4. Preparation of ionized mineral nanoparticles

     1) Scallop clam or cockleshell was calcined at 1,200 ° C for 2 hours, and the calcined mineral was pulverized into a pulverizer to obtain a powder, and a particle size of 30 to 300 mesh was obtained.

     2) The calcined mineral contained Mg, Na, K, P, S, Fe, and Zn as components. 25 W of ultrasonic waves was repeatedly applied to the mesh-sized mineral particles three times for 10 minutes, and 100 nm of the mineral particles obtained by pulverization were electrolyzed to obtain ionized mineral nanoparticles.

<Example>

To prepare a low pathogenic avian influenza virus disinfectant composition containing a giant mushroom extract according to the present invention, the composition components are uniformly blended in a composition ratio shown in [Table 2] below, followed by repeated addition of 25W ultrasonic waves 3 times for 10 minutes. , The mixture obtained by pulverization was electrolyzed again to obtain an ionized nano-mixture to obtain a disinfectant composition.

Composition and ratio of ingredients Ingredient Ingredient ratio (parts by weight) Purified water 95.5 Giant mushroom extract 6.0 Perilla leaf extract 3.0 Ionized mineral nanoparticles 2.0 Sodium cyanide 1.0 Glutaraldehyde 0.5 Guar hydroxypropyltrimonium chloride 0.5 Citric acid 0.5

<Experimental Example> Experimental method and evaluation of inhibitors

  1. Experimental method

    In order to test the disinfecting effect on the disinfectant composition of <Example>, the experiment was conducted according to National Veterinary Science and Quarantine Publication No. 2016-91 [Disinfectant Efficacy Test Guideline], and the results are shown in the [Table] below.

  2. Evaluation of inhibitors

   1) Each virus stock solution was diluted 1: 10 times, a 1.5 ml ep tube was used, and 1 μg / ml TPCK in DMEM was used as a dilution medium.

   2) For the reaction of the test substance and the virus, all test solutions and reagents were stored at 4 ° C to maintain the temperature, and an ep tube was used.

     2-1) 450 µl of test substance and 100 µl of virus were mixed.

     2-2) Substance control was mixed with 450 µl PBS and 100 µl virus.

     2-3) After mixing, the mixture was reacted at 4 ° C for 30 minutes, and mixed well every 10 minutes.

   3) 450 µl 1M Tris-HCl (pH 7.2) was added, and 1M Tris-HCl (pH 7.2) for neutralizing the test substance was used as a diluent.

   4) Virus titer was evaluated for the mixed solution, and a deep well plate was used.

      4-1) 10-fold dilution was performed at a ratio of 100 µl of the mixed solution and 900 µl of the medium.

4-2) After diluting multiples of 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , and 10 ^-6 , cells were inoculated with 100 µl and repeated 8 for each dilution.

       -Dilution medium: 1µg / ml TPCK in DMEM

     * In the case of fertilized eggs, 0.2 ml was inoculated into the allantoic cavity, cultured for 5 days in a 37 ° C. incubator, screened daily, and the presence or absence of virus growth was examined.

   5) The cells were reacted at 37 ° C for 1 hour.

   6) After the reaction, the inoculum was removed.

   7) 200 ml of culture medium was added.

     -Medium: 1 μg / ml TPCK in DMEM

8) Cultured in a 37 ° C. CO ₂ incubator, Carbapenemase production Enterobacteriaceae (CPE) was observed, and the results are shown in the [Table] below.

<Experimental Results> Evaluation of antiviral inhibitory ability against bird flu virus

From the above table, in case of pathogen control, the fertilized egg infection ability was confirmed even in the dilution factor 10 ⁸ or more. In addition, as criteria, for the disinfectant criteria 10 ^4. Suppressing ⁰ EID ₅₀ or more viral replication it was determined that the viral inhibitory effects. That is, it was confirmed that the example composition of the present invention inhibits virus proliferation of 10 ^4.0 EID ₅₀ or more virus.

Even in the evaluation of the inhibitory capacity using cells, it was confirmed that the example composition of the present invention inhibited the virus, but the influenza virus used in the test proliferated more significantly in the embryonated egg, and it was difficult to determine the correct inhibitory ability, but the virus compared to the overall virus control It was confirmed that the growth was inhibited.

<Comprehensive analysis of experimental results>

Summarizing the results of the experiment, the low-pathogenic bird flu virus disinfectant composition containing the extract of the king mushroom of the present invention not only has excellent inhibitory ability against avian influenza virus using embryonated eggs and cells, but also protects algae from disease caused by the virus, It was evaluated that it can be safely used without side effects on drugs and concerns about the safety of livestock products by residual drugs.

As described above, although the present invention has been described by a limited embodiment, the present invention is not limited thereto, and will be described below by the person skilled in the art to which the present invention pertains and the technical spirit of the present invention. Of course, various modifications and variations are possible within the scope of the claims.

Claims (15)

1) a first step of cutting a giant mushroom to an appropriate size, extracting using ethanol as a solvent, filtering and freeze-drying to obtain a giant mushroom extract;
2) a second step of cutting the perilla leaf to an appropriate size, extracting using ethanol as a solvent, filtering and freeze-drying to obtain a perilla leaf extract;
3) a third step of sintering shellfish, crushing, meshing, and then ultrasonically pulverizing to nano-particle, and then electrolyzing to ionize the mineral particles;
4) Purified water, the royal mushroom extract obtained in the first step, the perilla leaf extract obtained in the second step, sodium cyanate (NaOCN), glutaraldehyde (C ₅ H ₈ O ₂ ) in the ionizing mineral nanoparticles in the third step, A fourth step of making a mixed solution by mixing guar hydroxypropyltrimonium chloride and citric acid (C ₆ H ₈ O ₇ );
5) a method of manufacturing a low pathogenic avian influenza virus disinfectant composition comprising; a fifth step of nano-milling the mixed solution prepared in the fourth step so as not to be precipitated by ultrasonic grinding.
The method according to claim 1,
In the first step, ethanol was added at a weight ratio of 10 times to 70 Vol%, and at 80 ° C for 50 minutes, and at the second step, ethanol was added to 70% of the perilla leaf at a weight ratio of 10 times, respectively at 80 ° C for 30 minutes. A method of preparing a low pathogenic avian flu virus disinfectant composition that is refluxed.
The method according to claim 2,
In the third step, the shellfish is a scallop or cockleshell, and a method for preparing a low-pathogenic bird flu virus disinfectant composition.
The method according to claim 3,
In the third step, the firing is 2 hours at 1,200 ° C., and the pulverization is 30 to 300 mesh.
The method according to claim 4,
In the third step, the method of manufacturing the low-pathogenic avian flu virus disinfectant composition is to repeat the ultrasonic waves of 25W for 10 minutes and three times to make the particle size 100 nm. The method according to claim 5,
In the fourth step, the mixed solution is 95.5 parts by weight of purified water, 5.0-7.0 parts by weight of the king mushroom extract, 3.0 parts by weight of the perilla leaf extract, 2.0 parts by weight of ionized mineral nanoparticles, 1.0 parts by weight of sodium cyanate (NaOCN), glutaraldehyde (C ₅ H ₈ O ₂ ) 0.5 parts by weight, Guar Hydroxypropyltrimonium Chloride (Guar Hydroxypropyltrimonium Chloride) 0.5 parts by weight, citric acid (C ₆ H ₈ O ₇ ) 0.5 parts by weight of low pathogenic bird flu virus disinfectant composition Method of manufacturing.
The method according to claim 6,
The method of manufacturing a highly pathogenic avian influenza virus disinfectant composition in which the mixed solution is nano-sized by repeating 25W ultrasonic waves for 10 minutes and 3 times in the fifth step.
95.5 parts by weight of purified water, 5.0-7.0 parts by weight of Mushroom extract, 3.0 parts by weight of perilla leaf extract, 2.0 parts by weight of ionized mineral nanoparticles, 1.0 part by weight of sodium cyanide (NaOCN), glutaraldehyde (C ₅ H ₈ O ₂ ) 0.5 Low-pathogenic bird flu virus disinfectant composition containing 0.5 parts by weight of Guar Hydroxypropyltrimonium Chloride, 0.5 parts by weight of citric acid (C ₆ H ₈ O ₇ ).
The method according to claim 8,
The great mushroom extract is obtained by refluxing the mushrooms at 80 ℃ for 50 minutes with 70Vol% ethanol, a low pathogenic bird flu virus disinfectant composition.
The method according to claim 9,
The perilla leaf extract is a low pathogenic bird flu virus disinfectant composition obtained by refluxing perilla leaves with 70Vol% ethanol at 80 ° C for 30 minutes.
The method according to claim 10,
The ionized mineral nanoparticles are low pathogenic bird flu virus disinfectant composition obtained by calcining scallop or cockleshell at 1,200 ° C for 2 hours.
The method according to claim 11,
The ionized mineral nanoparticles are low pathogenic avian flu virus disinfectant composition containing Mg, Na, K, P, S, Fe, and Zn as constituents.
The method according to claim 12,
The low pathogenic avian influenza virus is H9N2 low pathogenic avian influenza virus disinfectant composition.
The method according to any one of claims 8 to 13,
The low pathogenic avian influenza virus disinfectant composition further comprises at least one additive selected from a dispersion and a carrier.
An antiviral cleaning tissue, characterized in that it is prepared by impregnating the low pathogenic avian influenza virus disinfectant composition of claim 8.