WO2010024598A2

WO2010024598A2 - Method for preparing antimicrobial, antifungal, and antiviral compositions

Info

Publication number: WO2010024598A2
Application number: PCT/KR2009/004779
Authority: WO
Inventors: 조일훈; 박동아; 양영열
Original assignee: 주식회사 지피엔이
Priority date: 2008-08-27
Filing date: 2009-08-27
Publication date: 2010-03-04
Also published as: WO2010024598A3

Abstract

The present invention relates to a method for preparing antimicrobial, antifungal, and antiviral compositions. In detail, the present invention relates to a method for preparing antimicrobial, antifungal, and antiviral compositions including the steps of: i) reacting a first alcohol containing at least one dispersion stabilizer and a silver precursor to prepare a silver colloid solution; and ii) adding a titanium precursor, a second alcohol, and an acid catalyst to the obtained solution by diluting the silver colloid solution with water to generate titanium dioxide nanoparticles in the silver colloid solution. The compositions of the present invention contain 0.0000005 to 10 weight parts of the silver colloid, 0.0000001 to 15 weight parts of the titanium dioxide nanoparticles, 0.00001 to 6 weight parts of at least one dispersion stabilizer, and 55 to 99 weight parts of water. The present invention also relates to a method for destroying the new H1N1 virus by using the compositions.

Description

Method for preparing antimicrobial, antifungal and antiviral compositions

The present invention relates to methods of making antimicrobial, antifungal and antiviral compositions. More specifically, the present invention comprises the steps of i) preparing a silver colloidal solution by reacting a silver precursor with a first alcohol containing at least one dispersion stabilizer; ii) adding a titanium precursor, a second alcohol and an acid catalyst to a solution of diluting the silver colloidal solution with water to produce titanium dioxide nanoparticles in the silver colloidal solution, wherein 0.0000005 parts by weight of the silver colloid To 10 parts by weight, 0.0000001 parts by weight to 15 parts by weight of the titanium dioxide nanoparticles, 0.00001 parts by weight to 6 parts by weight of the at least one dispersion stabilizer, and 55 parts by weight to 99 parts by weight of water, antibacterial, A method for preparing antifungal and antiviral compositions and a method for killing influenza A (H1N1) virus using the composition.

Silver has long been known to have antimicrobial effects. Particularly, silver colloid refers to a substance dispersed in an aqueous solution in a clustered state (10 nm to 150 nm in size). It has been shown to have strong antibacterial activity against harmful bacteria and fungi. It is also observed that these silver colloidal particles do not kill most of the beneficial bacteria when applied to the human body.

Moreover, in the United States, silver colloidal solution is recognized as a natural substance harmless to the human body registered in the FDA pharmaceutical range, and recently, it is known as a nontoxic substance to human or living body enough to be recognized as a food additive as a nontoxic preservative.

As described above, the silver colloidal solution has been proved to be very excellent in antibacterial and non-toxic to human body. Currently, in the world, the manufacture of silver particles more effective for antibacterial and eradication and treatment of pathogenic bacteria using the same, cosmetics, textiles, wallpaper, washing machine Research has been conducted to utilize silver in various products such as clothing and clothing.

Photocatalysts generate active oxygen and hydroxyl radicals (OH radicals) when they receive light with more than the appropriate energy. They exhibit decomposing and antimicrobial effects of odorous substances by their strong oxidation and reduction. It is called. A semiconductor is excited from the valence band to the conduction band when a certain area of energy is applied. At this time, electrons ( e − ) are formed in the conduction band, and holes (h +) are formed in the valence electron band. The electrons and holes thus formed cause various reactions such as decomposing harmful substances by strong oxidation or reduction.

As such, titanium dioxide (TiO ₂ ) is most widely used because of its chemical stability and excellent advantages as a semiconductor among various oxide materials having photocatalyst properties. In the case of titanium dioxide, when it receives an energy of about 3.0 eV (the atom corresponds to light of 380 nm wavelength in the energy gap between the electron band and the conduction band), it fully functions as a photocatalyst.

The light hits the photocatalyst is titanium dioxide electrons generated (e-) and holes (h +) is a superoxide anion on the titanium dioxide surface causes the O _{_2,} H ₂ O and the reaction of each air (O ₂ ^-) and hydroxyl radical It produces two kinds of active oxygen which are (.OH). In particular, since the hydroxyl radical has a high redox potential, it is excellent for purifying NOx, volatile organic compounds (VOCs) and various odors. Not only can it be completely removed, but it also has the ability to oxidize all the target substances such as pathogenic E. coli, Staphylococcus aureus, O-157, etc.

Such titanium dioxide is also reacted by solar energy or fluorescent light, and is economical because it exhibits a permanent function by the cycle of 'fixing to an object → photolysis → regeneration'. In addition, the by-products after the reaction is a material that is harmless to humans and the environment with water and CO ₂ , and can be applied to various fields.

SARS is likely to be a variant of the existing corona virus that causes colds, according to the World Health Organization. Corona viruses not only have a high incidence of mutations specific to RNA viruses, but also have a high recombination frequency as RNA polymerases tend to jump from place to place as they replicate genomes.

Corona virus causes respiratory symptoms such as colds when it is infected with humans, but it has not been a major problem since it is not high in risk. However, some animals, such as cows, dogs, pigs, and birds, have sometimes acted as deadly pathogens, which is why infectious disease experts estimate that the corona virus has been transformed into livestock, such as pigs and chickens, into human bodies, causing deadly diseases. . SARS spreads the virus as it moves into the respiratory tract after inhaling “air droplets,” which are protruding drops when the patient coughs, or touching objects (such as door handles or telephone computer keyboards) used by the patient.

Human influenza (HI), a pandemic flu that is popular during the transitional season and in winter (usually from November to March), is a communicable disease caused by the influenza virus, unlike the common cold. Influenza A, B, and C are single-stranded RNAs of the orthomyxovirus family. In particular, influenza A has frequent nuclear rearrangements, causing pandemic and pandemic, and influenza C only causes minor respiratory diseases.

Avian influenza (AI) is a virus that occurs in poultry or wild birds such as chickens and ducks. Influenza viruses are generally classified into types A, B, and C. Among them, A and B are known to cause human infection, and only A type is known to cause pandemic.

There are two proteins on the surface of the influenza virus: hemagglutinin (HA) and neuraminidase (NA) .There are 16 types of HA and 9 types of NA. Influenza virus of the kind (= 16x9) will exist. Among them, three types of HA (H1, H2, H3) and two types of NA (N1 and N2) are generally reported as a type of virus causing human influenza infection, and influenza infection of birds is mainly H5 type. Or H7 type.

And among these, the H5N1 virus is highly pathogenic and is known to be the cause of the poultry avian influenza outbreak as we know it. Avian influenza spreads in the form of virus-infested bird runny nose, respiratory secretions, and birds in contact with feces again, especially feces contaminated with influenza cause infection through the oral cavity. Therefore, instruments, media, feed, cages and clothes contaminated with respiratory secretions or feces of birds play an important role in the spread of avian influenza.

Prior arts for sterilization and sterilization by corona virus and influenza virus inhibitors, silver or titanium dioxide photocatalysts are as follows. Currently, products having inhibitory ability against coronaviruses are mainly substances extracted from synthetic organic matter or natural products (Korean Unexamined Patent Publication No. 2003-0063961 and Japanese Unexamined Patent Publication No. 2000-44473), and are not inorganic solutions.

The novel influenza A (H1N1) virus, which has recently caused respiratory infections in humans around the world, began spreading humans in Mexico and the United States in early April 2009.

Influenza A (H1N1) virus infection has been reported to be possible up to seven days after symptom onset, and in children, this period may be longer. To prevent this, it is important to wash your hands. The door handles and payphones that we touch unintentionally can also contain viruses, so wash your hands frequently and avoid touching your eyes, nose, and mouth while you are away.

Prior arts for sterilization and sterilization by corona virus and influenza virus inhibitors, silver or titanium dioxide photocatalysts are as follows.

Currently, products having inhibitory ability against coronaviruses are mainly substances extracted from synthetic organic matter or natural products (Korean Unexamined Patent Publication No. 2003-0063961 and Japanese Unexamined Patent Publication No. 2000-44473), and are not inorganic solutions.

Antimicrobial products using silver colloid or titanium dioxide photocatalyst alone as inorganic materials are based on the above known theory. Various products such as silver coated clothing, antibacterial spray (Korea Patent Publication No. 2002-0008375), filter coating agent for air purifier, etc. When silver is used, there is almost no change in performance according to ambient conditions, but a product using only a photocatalyst has a big disadvantage in that it does not properly display performance in an environment in which light is insufficient.

In addition, as a technology for a product and a method for producing antimicrobial properties using silver and titanium dioxide, Korean Patent Publication No. 1998-0007982 (Method for preparing inorganic antimicrobial agent), 2001-0057595 (Method for preparing photocatalyst coated with silver) And 2003-0037050 (titanium dioxide photocatalyst containing an antibacterial metal component and a method for producing the same).

The Patent Publication No. 2003-0037050 discloses an antimicrobial metal such as silver in the form of a colloid, mixed with titanium dioxide, a hydrothermal synthesis process to produce a crystalline oxide, and their surface is coated with a sol-gel method to obtain micro A photocatalytic material containing an antibacterial metal is made through a complex process of encapsulation. No. 1998-0007982 mixes phosphoric acid, silver nitrate, titanium dioxide powder and colloidal silica, and then dry and calcinates them to carry the silver ions and simultaneously carry the silver ions. One inorganic antimicrobial agent is prepared, and No. 2001-0057595 relates to a method for producing a fine powder photocatalyst in the form of applying silver to the outside of the photocatalyst powder by substituting silver to the photocatalyst.

However, the composition according to the present invention and a method for synthesizing the same are different from those of the conventional silver and titanium dioxide, which exhibit antimicrobial properties and manufacturing methods.

The inventors of the present invention, in order to overcome the problems of the prior art described above, two nanomaterials having antimicrobial, antifungal and antiviral functions, silver colloid and titanium dioxide, a simple mixture such as the above patents, composite hydrothermal synthesis, and intermetallic substitution By diluting a solution in which nano-sized colloids of silver are uniformly dispersed without distillation, calcination, etc. with distilled water to an appropriate ratio (10 ppm to 5,000 ppm), and then using it as a solvent for synthesis with titanium dioxide The present invention has been completed.

In addition, the synergistic effect of silver and titanium dioxide is shown in the antimicrobial / antifungal and antiviral ability as well as the simplicity of synthesis. In particular, the inorganic solution prepared according to the present invention is synthesized with colloidal and titanium dioxide particles, and exhibits strong antibacterial, antifungal and antiviral ability even in the presence or absence of light.

The solution prepared according to the present invention not only removes various existing viruses, bacteria, fungi and odors, but also coronaviruses (TGEV, PEDV) and avian influenza (AI) virus, swine influenza (SI) virus, which in particular cause SARS, and It has a potent antibacterial / antifungal and antiviral activity of at least 99.9% against human influenza (HI) virus.

The basic object of the present invention is i) preparing a silver colloidal solution by reacting a silver precursor with a first alcohol containing at least one dispersion stabilizer; ii) adding a titanium precursor, a second alcohol and an acid catalyst to a solution of diluting the silver colloidal solution with water to produce titanium dioxide nanoparticles in the silver colloidal solution, wherein 0.0000005 parts by weight of the silver colloid To 10 parts by weight, 0.0000001 parts by weight to 15 parts by weight of the titanium dioxide nanoparticles, 0.00001 parts by weight to 6 parts by weight of the at least one dispersion stabilizer, and 55 parts by weight to 99 parts by weight of water, antibacterial, It is to provide an antifungal and antiviral composition.

Still another object of the present invention is 0.0000005 to 10 parts by weight of silver colloidal particles, 0.0000001 to 15 parts by weight of titanium dioxide nanoparticles, 0.00001 to 6 parts by weight of at least one dispersion stabilizer, and 55 to 99 parts by weight It is to provide a composition for killing influenza A (H1N1) virus, which contains negative water.

Still another object of the present invention is 0.0000005 to 10 parts by weight of silver colloidal particles, 0.0000001 to 15 parts by weight of titanium dioxide nanoparticles, 0.00001 to 6 parts by weight of at least one dispersion stabilizer, and 55 to 99 parts by weight It provides a method for killing a new influenza A (H1N1) virus, comprising the step of applying a composition for killing the new influenza A (H1N1) virus containing the water.

Still another object of the present invention is to provide a novel influenza A (H1N1) virus blocking filter coated with a composition for killing influenza A (H1N1) virus including silver colloid particles, titanium dioxide nanoparticles, and at least one dispersion stabilizer. To provide.

The basic object of the present invention described above is i) preparing a silver colloid solution by reacting a silver precursor with a first alcohol containing at least one dispersion stabilizer; ii) adding a titanium precursor, a second alcohol and an acid catalyst to a solution of diluting the silver colloidal solution with water to produce titanium dioxide nanoparticles in the silver colloidal solution, wherein 0.0000005 parts by weight of the silver colloid To 10 parts by weight, 0.0000001 parts by weight to 15 parts by weight of the titanium dioxide nanoparticles, 0.00001 parts by weight to 6 parts by weight of the at least one dispersion stabilizer, and 55 parts by weight to 99 parts by weight of water, antibacterial, It can be achieved by providing a method for preparing antifungal and antiviral compositions.

When nano-sized particles are present in a high concentration in the solution, the particles are agglomerated with each other by the self-association reaction, and thus, it is difficult to maintain homogeneity due to precipitation due to an increase in particle diameter. Therefore, in the production method of the present invention to prepare a nano-sized silver particle colloid in order to eliminate such disadvantages, the prepared silver colloidal solution and nano-sized titanium dioxide solution is synthesized through hydrolysis and condensation reaction.

The silver colloidal particles prepared in step i) of the method for preparing a composition of the present invention have an average particle diameter of 1 nm to 100 nm, and a silver colloidal solution having a concentration of about 50,000 ppm is prepared. Preferably, the silver colloidal solution is diluted with water to a concentration of 10 ppm to 5,000 ppm and used in step ii).

In addition, the silver precursor used in step i) is preferably silver nitrate (AgNO ₃ ) or silver acetate (CH ₃ COOAg).

In addition, the first alcohol of step i) is preferably selected from methanol, ethanol, isopropyl alcohol or butanol.

In addition, the dispersion stabilizer of step i) is lysolecithin, flavonoid, flavonoid, Tween 20, tween 40, tween 80, polyvinylpyrrolidone and polyvinyl alcohol (polyvinylalcohol) It is preferably at least one selected from the group consisting of.

In addition, the reaction temperature and reaction time of step i) is preferably 70 ℃ to 90 ℃ and 2 hours to 5 hours, respectively.

The titanium precursor of step ii) of the method of preparing a composition of the present invention is preferably selected from the group consisting of titanium tetraisopropoxide (TTIP), titaniumethoxyorthotitanate (TEOT), and titaniumbutoxyorthotitanate (TBOT).

In addition, the second alcohol of step ii) is preferably selected from the group consisting of isopropyl alcohol, ethanol and butanol.

In addition, the acid catalyst of step ii) is preferably selected from the group consisting of nitric acid, hydrochloric acid, sulfuric acid and oxalic acid.

In addition, the reaction temperature and the reaction time of the step ii) is preferably 60 ℃ to 85 ℃ and 4 hours to 8 hours, respectively.

The size of the titanium dioxide nanoparticles prepared according to the method for producing a composition of the present invention is preferably 1 nm to 100 nm.

The composition prepared according to the above production method contains 1 wt% to 5 wt% of titanium dioxide particles having an average particle diameter of milky white of 1 nm to 100 nm. In general, when titanium dioxide is prepared in a silver colloidal solution, when the concentration of the silver colloid is higher than a certain value or when the concentration of the titanium dioxide raw material used in the sol-gel reaction is high, the stability of the silver colloidal titanium dioxide is very poor. Precipitation occurs.

According to the method of the present invention, the silver colloid particle concentration of 10ppm to 5,000ppm, titanium dioxide concentration 0.01wt% to 5wt% can be sufficiently controlled within the limit to achieve homogeneous dispersion without aggregation.

In addition, the composition prepared according to the method of the present invention is preferably used after neutralizing with a base.

As a result of analyzing the composition prepared by the method of the present invention through the TEM photograph of Figure 1, it formed a uniform particle size and uniform distribution of 20nm or less, and also excellent antibacterial, antifungal and antiviral activity. 2 is different from the pattern of silver colloid, titanium dioxide and “silver colloid / titanium dioxide-mixing” through XRD analysis, the composition prepared by the method of the present invention has a structure in which silver colloid and titanium dioxide are ideally bonded to each other. Indicates.

The antimicrobial mechanism of the composition prepared by the method of the present invention can be expressed in two ways. First, viruses, bacteria, and fungi interfere with the function of the absorption system (Coenzyme A), which is necessary for oxygen and digestive metabolism (nanoparticles bind to the virus and coagulate), suffocating or assuring, ie nanoparticles. Binds to proteins such as cell membranes and enzymes and inhibits cell metabolism. Second, the nanoparticles penetrate into the virus / bacteria body and destroy and remove the organic structure of the cell, and the nanocomposite destroys the cell membrane by binding strongly and momentarily with the microorganism's -SH, -COOH, and -OH groups. Plays a role in disturbing the cells.

As described above, the composition prepared according to the method of the present invention can be sprayed by diluting with distilled water, alcohol, etc., depending on the application object, and prepared by spraying a sprayable solution, vial, spray, aerosol, or the like. Or by applying or impregnated with natural fibers, nonwovens, masks, filters, etc. can be applied to antibacterial and anti-rust products.

Another object of the present invention described above is 0.0000005 parts by weight to 10 parts by weight of silver colloidal particles, 0.0000001 parts by weight to 15 parts by weight of titanium dioxide nanoparticles, 0.00001 parts by weight to 6 parts by weight of at least one dispersion stabilizer, and 55 parts by weight to It can be achieved by providing a composition for killing influenza A (H1N1) virus, comprising 99 parts by weight of water.

Dispersion stabilizers of the composition for killing influenza A (H1N1) virus of the present invention are lysolecithin, flavonoid, tween 20, tween 40, tween 80, polyvinylpyrrolidone and It is preferably at least one selected from the group consisting of polyvinyl alcohol.

In addition, the size of the silver colloidal particles and titanium dioxide nanoparticles of the new influenza A (H1N1) virus killing composition of the present invention is preferably 1 nm to 100 nm.

Another object of the present invention described above is 0.0000005 parts by weight to 10 parts by weight of silver colloidal particles, 0.0000001 parts by weight to 15 parts by weight of titanium dioxide nanoparticles, 0.00001 parts by weight to 6 parts by weight of at least one dispersion stabilizer, and 55 parts by weight to It can be achieved by providing a method for killing a new influenza A (H1N1) virus, comprising applying a composition for killing a new influenza A (H1N1) virus comprising 99 parts by weight of water.

The composition for killing influenza A (H1N1) virus of the present invention effectively kills the influenza A (H1N1) virus, as described in the following examples. Therefore, it is possible to control and remove the H1N1 virus by applying the H1N1 virus composition as necessary.

Dispersion stabilizers used in the method of killing the new influenza A (H1N1) virus of the present invention are lysolecithin, flavonoid, tween 20, tween 40, tween 80, polyvinylpyrrolidone ( It is preferably at least one selected from the group consisting of polyvinylpyrrolidone) and polyvinyl alcohol.

In addition, the size of the silver colloidal particles and titanium dioxide nanoparticles used in the method of killing the influenza A (H1N1) virus is preferably 1 nm to 100 nm, respectively.

Another object of the present invention described above is a novel influenza comprising 0.0000005 parts by weight to 10 parts by weight of silver colloidal particles, 0.0000001 parts by weight to 15 parts by weight of titanium dioxide nanoparticles, and 0.000005 parts by weight to 6 parts by weight of at least one dispersion stabilizer. A (H1N1) virus killing composition can be achieved by providing a filter for the new influenza A (H1N1) virus blocking.

Dispersion stabilizers used in the novel influenza A (H1N1) virus blocking filter of the present invention is lysolecithin, flavonoids, tween 20, tween 40, tween 80, polyvinylpyrrolidone ) And polyvinyl alcohol (polyvinylalcohol) is preferably any one or more selected from the group consisting of.

In addition, the size of the silver colloidal particles and the titanium dioxide nanoparticles used in the new influenza A (H1N1) virus blocking filter is preferably 1 nm to 100 nm.

The composition of the present invention is characterized in that it can exert a synergistic effect to enhance the strong antibacterial and antiviral ability by nano silver particles and photocatalyst particles even in the presence or absence of light, and volatility due to the unique organic oxidation ability It also possesses the decomposition function of organic substances and odorous substances at the same time and is easy to use because it is manufactured in a sprayable form.

Due to the above characteristics, the composition of the present invention is a strong antibacterial and odorous substance against various viruses, bacteria and fungi, including varietal coronavirus, swine epidemic diarrheal virus, swine infectious gastroenteritis virus, E. coli, influenza, etc. It has a wide range of uses for the purpose of simultaneously deodorizing, decomposing trace volatile organic substances and obtaining the purpose of fragrance.

In particular, the composition of the present invention can be used for the prevention and killing of H1N1 virus because of its excellent killing effect against H1N1 virus. In addition, the swine flu can be effectively prevented through a mask using a filter coated with the composition of the present invention.

1 is a TEM photograph of a composition prepared according to the method of the present invention.

2 is XRD results for silver colloidal particles, titanium dioxide nanoparticles, a mixture of silver colloidal particles and titanium dioxide nanoparticles, and a composition prepared according to the method of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples or drawings. However, the following description of the embodiments or drawings is only intended to specifically describe the specific embodiments of the present invention, it is not intended to limit or limit the scope of the present invention to the contents described therein.

Antibacterial activity test against Staphylococcus aureus, bactericidal test against E. coli, antifungal test, antiviral test against SARS coronavirus and influenza virus, oral toxicity and skin irritation of the composition prepared according to the method of the present invention Each test method is as follows.

end. Test method for antibacterial activity against Staphylococcus aureus

1. Inoculate a single colony of test strain into 5 ml of LB broth medium (10 g of peptone, 5 g of yeast extract, 10 g of NaCl, 15-20 g / L of agar), and overnight at 37 ° C. Incubated.

2. Turbidity was adjusted to 0.5 by measuring O.D. (optical density) at 660 nm.

3. The test solution adjusted to 0.5 was inoculated in 5 ml of LB broth for 100-fold dilution.

4. Sample processing is as follows.

Treatment Concentration: Samples were treated so that the final concentration was 10 ³ fold dilution.

Treatment time: 1 hour and 3 hours of incubation of the sample, and diluted (100 times, 10,000 times), 100 μl was applied to the LB plate (LB broth + agar 15 ~ 20g / L) and overnight at 37 ℃ Incubated for

5. The number of colonies was measured and the antimicrobial activity was measured compared to the number of colonies of the control group.

I. Test method for antibacterial activity against Escherichia coli

After spreading E. coli (Escherichia coli) to the medium, the solution of the present invention diluted 103 times in distilled water is sprayed three times (about 0.5 ml) each. The treated medium was incubated in an incubator equipped with three fluorescent lamps for 24 hours, and then bactericidal power was examined by measuring colony numbers.

All. Antifungal Test Method for Five Species

The antifungal test for the composition according to the present invention was carried out according to ASTM G 21-96 (2002). ATCC 15233). Test temperature and test period are as follows.

1.Test temperature: 28 ± 1 ℃

2. Test period: 4 weeks

la. Test method for antiviral activity against coronavirus

As a virus to measure antiviral activity for the composition according to the present invention, PEDV (Porcine Epidemic Diarrhea Virus) and TGEV (Transmissible GastroEnteric Virus) were selected. PEDV is a coronavirus that proliferates in the monkey kidney cell line Vero cells, such as the animal cell line known as SARS virus (VeroE6), and the VeroE6 cell line is a derivative cell line of Vero cell line. Test methods, data processing and evaluation methods are as follows.

1. Vero cells, the host cells of the virus, were placed in each well of a 96 well plate, and 2 × 10 ⁴ cells were incubated for 16 hours to completely cover the cells with a monolayer on the bottom of each well.

2. The composition according to the present invention was diluted 10-fold continuously in distilled water, and the virus solution diluted to a constant concentration was mixed and left at 4 ° C. for 30 minutes, and then inoculated into each well of a plate in which each cell was cultured.

3. As a control, a culture medium containing no composition according to the present invention, a culture containing only virus, and a well containing only a composition according to the present invention and containing no virus were used.

4. Test 96 well plates were terminated after 40 hours and the cells in 96 well plates were fixed with 70% acetone solution and completely dried.

5. The assay was performed by staining the cell proteins remaining in each well with SRB (0.4% Sulforhodamine B in 1% acetic acid) and re-dissolving the absorbance of each well using an EIA reader (96 well plate reader). The ratio of the treated group to the untreated control group was analyzed.

6. Each test was repeated three times.

7. Of group (A) not treated with virus, group (B) treated with composition according to the present invention, group (C) treated with virus only and group (D) treated with virus and composition according to the present invention together Based on the absorbance value, antiviral activity was calculated by the following equation to evaluate the overall performance of the composition according to the present invention.

% Antiviral activity = (D-C) / (B-C) x 100

hemp. Antiviral test method for influenza virus

Human Influenza (HI) Virus

In vitro (In vitroAntiviral function in

Condition: Contact time 1 hour

Plaque assay: Plaque forming unit (pfu) using MDCK cells

Calculation

1) Influenza A virus (H3N2)

Test Virus: A / Shangdong / 9/93 EC ₅₀ : 0.02-0.04%

2) Influenza A virus (H1N1)

Test Virus: A / Bayern / 7/95 EC ₅₀ : 0.08-0.16%

A / PR / 8/34 EC ₅₀ : 0.04-0.08%

3) Influenza B virus

Test Virus: B / Yamagata / 16/88 EC ₅₀ : 0.02-0.04%

4) 50% inactivation time: within 1-5 minutes after contact with 1% solution

Within 1 hour after contact with 0.05% solution

2. Avian Influenza (AI) Virus

Validation of antiviral function in vivo

Condition: 1 hour contact time

By: whether the embryos infected with the virus grown (embryo infection index calculated EC ₅₀₎

Test Virus: H9N2 Avian Influenza EC ₅₀ : 0.02-0.04%

bar. How to measure inactivation against the H1N1 virus

1.Method for measuring inactivation of the swine flu virus of the composition of the present invention

1) Dilute the composition of the present invention 10 times with PBS pH 7.4.

2) Apply 100 μl of H1N1 virus of 10 ⁶ EID ₅₀ / ml to the diluted composition (EID ₅₀ = 50% egg infective dose).

3) React at room temperature for 20 minutes.

4) After reacting for 20 minutes, dilute serially 10 times with PBS.

5) Inoculate 10-fold diluted eggs into 10-day-old eggs.

6) The inoculated fertilized eggs are incubated in an incubator at 35 ° C. for 48 hours and then refrigerated for 4 hours in a refrigerator at 4 ° C.

7) The presence of virus in the ureters of chilled fertilized eggs is measured by hemagglutination, and 1og ₁₀ EID ₅₀ / ml is measured.

2. Method for measuring inactivation ability against the swine flu virus of a filter treated with the composition of the present invention

1) The filter treated or untreated filter of the composition of the present invention is cut into about 2 cm X 2 cm and placed in a petri dish.

2) Apply 100 ml of the H1N1 virus to 10 ⁶ EID ₅₀ / ml on the cut filter.

3) React the virus-filtered filter at the laboratory temperature for 20 minutes.

4) After reacting for 20 minutes, wash the filter with 1 ml of PBS (pH 7.4) and dilute serially 10 times with PBS.

5) Inoculate 10-fold dilutions into 10-day-old fertilized eggs.

6) The inoculated fertilized eggs are incubated in an incubator at 35 ° C. for 48 hours and then refrigerated in a refrigerator at 4 ° C. for 4 hours.

four. Safety test for oral toxicity and skin irritation

As part of the safety test for the composition according to the present invention, oral toxicity test and skin irritation test were conducted in accordance with Korean Food Safety Agency No. 1999-61.

Example 1 Synthesis of Compositions According to the Present Invention

First, silver colloid was prepared by using 10 g of silver nitrate (AgNO ₃ ), 20 g of ethanol, and 0.01 g of flavonoid (flavonoid) as a dispersion stabilizer at 80 ° C. for 5 hours to prepare silver colloidal particles by alcohol reduction. At this time, the silver colloidal solution prepared was diluted with distilled water so that the average particle diameter was 20-50 nm in size and a 50,000 ppm silver colloidal solution was prepared to have a concentration of 10-5,000 ppm.

Subsequently, a silver colloidal solution diluted to 500 ppm with water was used as a solvent, and a solution of 35 g of titaniumethoxyorthotitanate (TEOT) and ethanol was added thereto. Was performed. At this time, 0.5 g of HNO ₃ was used as a catalyst for the hydrolysis reaction. The reaction temperature was heated to 60 to 85 ℃ and stirred for 4 to 8 hours to synthesize a composition according to the present invention containing titanium dioxide particles having an average particle diameter of 1 to 100 nm in milky white at a concentration of 1wt% to 5wt%.

Examples 2-7.

As shown in Table 1, the composition according to the present invention was synthesized in the same manner as in Example 1 with only the addition amounts of silver nitrate and TEOT added.

Table 1

	Silver Nitrate Content (g)	Ethanol Content (g)	Flavonoid Content (g)	TEOT Content (g)	Nitric acid content (g)	stability
Example 1	10	20	0.01	35	0.5	○
Example 2	5	20	0.01	35	0.5	○
Example 3	15	20	0.01	35	0.5	○
Example 4	20	20	0.01	35	0.5	×
Example 5	10	20	0.01	20	0.5	○
Example 6	10	20	0.01	40	0.5	○
Example 7	10	20	0.01	50	0.5	×

○: Synthesis of colloid of stable silver-titanium dioxide without precipitation occurs at all

×: precipitation immediately after synthesis

end. Antibacterial Activity Test Results for Staphylococcus Aureus

The antimicrobial activity of the composition prepared by the method of the present invention and the solution prepared by the conventional mixing method showed that the removal rate for Staphylococcus aureus was 100% and 63% after 1 hour of treatment time. The prepared composition showed more excellent antimicrobial activity results (Table 2).

TABLE 2

Comparison of Antimicrobial Activity Results against Staphylococcus Aureus of a Solution Prepared by the Method of the Present Invention and the Conventional Mixing Method (cfu / plate)

Staphylococcus aureus	Silver colloid / titanium dioxide solution prepared by the method of the present invention		Solution prepared by conventional mixing method ^*
	Dilute OD = 0.5 to 1/100		Dilute OD = 0.5 to 1/100
	1 hours	3 hours	1 hours	3 hours
	10 ^-4 dilution	10 ^-4 dilution	10 ^-4 dilution	10 ^-4 dilution
Control	About 1,000	Innumerable	582	Innumerable
Residual bacteria count / removal rate (%)	0/100	0/100	216/63	280 /-

*: The conventional mixing method is a solution in which the silver colloid and the titanium dioxide solution are prepared, and then the titanium dioxide solution is simply mixed with the silver colloid solution. At this time, the mixing ratio of silver colloid solution: titanium dioxide solution was 8: 2.

I. Antibacterial activity test results for E. coli

The composition according to the present invention eliminated up to 99.999% E. coli.

All. Antifungal Test Results of Five Species

The results of “grade 0” showed no mold growth at all for five fungal strains (Table 3).

TABLE 3

Used strain	Aspergillus niger ATCC 9642 Chatomium globosum ATCC 6205 Penicillium pinophilum ATCC 11787 Gliocladium virens ATCC 9645 Aureobasidium pullulans ATCC 15233	ASTM G 21-96 (2002)
ranking	0	ASTM G 21-96 (2002)

la. Antiviral test results for coronavirus

Three replicates of antiviral activity revealed similar types. Within this dilution range, there was little cytotoxicity by the composition. When the composition was diluted 100-fold, antiviral activity against PEDV (Porcine Epidemic Diarrhea Virus) and TGEV (Transmissible Gastroenteric Virus) was 99.99% and 99.9%, respectively. In the case of diluting 1,000-fold, 99.9% and 93.0% or more showed more than 90% virus inhibition. In general, the higher the concentration of the composition, the higher the inhibitory activity against the virus (Table 4).

Table 4

Test Methods	Composition according to the present invention
Active test	Efficacy (100-fold dilution)		Efficacy (1,000-fold dilution)
Active test	PEDV	TGEV	PEDV	TGEV
Antiviral activity	99.99% or more	99.99% or more	99.99% or more	93.0% or more

hemp. Results of antiviral activity test against influenza virus in the composition

According to the antiviral activity test method for influenza virus of the composition according to the present invention, the effect was demonstrated for human influenza virus and avian influenza virus (Table 5).

Table 5

Kinds	Human influenza virus; A type: H1N1, A type: H3N2, B type	Avian influenza virus; H9N2
result	EC ₅₀ : 0.02-0.04%	EC ₅₀ : 0.02-0.04%

bar. Inactivation test results for swine flu (H1N1) virus of the composition of the present invention

The composition according to the present invention was tested according to the test for inactivation against the swine flu virus, and the composition was 100% free of the swine flu virus. In addition, the filter treated with the composition removed more than 99.99% of swine flu virus (Table 6)

Table 6

sample	Virus titer (log ₁₀ EID ₅₀ / ml)
Compositions of the Invention	0	0	0	0	0
Filter Treated with Composition of the Invention	<1.0	<1.0	<1.0	<1.0	<1.0
Filter not treated with the composition of the present invention	6.0	6.5	6.5	6.0	6.0
Virus control	6.5	6.0	6.5	6.5	6.0

four. Safety test results for oral toxicity and skin irritation of the composition according to the present invention

As a safety test for the composition according to the present invention, the oral toxicity test and the skin irritation test based on the Korean Food Safety Agency Notification No. 1999-61 are shown in Table 7.

TABLE 7

Test Items	Test result	Test Methods
Oral Toxicity	After the oral administration of the test substance to the rat at a concentration of 5,000 mg / kg BW, the number of dead animals, general symptoms, weight change and autopsy findings were observed for 14 days. The general symptoms and autopsy findings related to the administration of the test substance were recognized. No deaths were observed, and no significant weight changes were recognized. Therefore, the approximate lethal dose for the composition is judged to be more than 5,000 mg / kg BW for both male and female.	KFDA Notification No. 1999-61
Skin irritation	The skin application of the composition in New Zealand White rabbits does not cause erythema, skin and swelling, so the primary skin irritation index PII (Primary Irritation Index) value is calculated as “0.0”, resulting in non-irritating substances. Was evaluated. The above results confirmed that the composition is a harmless substance when applied to humans or animals.	KFDA Notification No. 1999-61

Claims

i) preparing a silver colloidal solution by reacting a first alcohol containing at least one dispersion stabilizer with a silver precursor;

ii) adding titanium precursor, a second alcohol and an acid catalyst to a solution of diluting the silver colloidal solution with water to produce titanium dioxide nanoparticles in the silver colloidal solution;

0.0000005 to 10 parts by weight of the silver colloid, 0.0000001 to 15 parts by weight of the titanium dioxide nanoparticles, 0.00001 to 6 parts by weight of the at least one dispersion stabilizer, and 55 to 99 parts by weight of the water. A method for producing an antibacterial, antifungal and antiviral composition, containing by weight.
The method of claim 1, wherein the silver precursor of step i) is silver nitrate (AgNO 3 ) or silver acetate (CH 3 COOAg).
The method of claim 1, wherein the first alcohol of step i) is selected from the group consisting of methanol, ethanol, isopropyl alcohol and butanol.
The method of claim 1, wherein the dispersion stabilizer of step i) is lysolecithin, flavonoids, Tween 20, Tween 40, Tween 80, polyvinylpyrrolidone and polyvinyl alcohol (polyvinylalcohol), characterized in that any one or more selected from the group consisting of, antimicrobial, antifungal and antiviral composition production method.
The method of claim 1, wherein the titanium precursor of step ii) is selected from the group consisting of titanium tetraisopropoxide (TTIP), titaniumethoxyorthotitanate (TEOT), and titaniumbutoxyorthotitanate (TBOT).
The method of claim 1, wherein the second alcohol of step ii) is selected from the group consisting of isopropyl alcohol, ethanol and butanol.
The method of claim 1, wherein the acid catalyst of step ii) is selected from the group consisting of nitric acid, hydrochloric acid, sulfuric acid and oxalic acid.
According to claim 1, wherein the reaction temperature and reaction time of step i) is characterized in that 70 ℃ to 90 ℃ and 2 hours to 5 hours, respectively, antimicrobial, antifungal and antiviral composition production method.
The method of claim 1, wherein the reaction temperature and reaction time of step ii) are 60 ° C to 85 ° C and 4 hours to 8 hours, respectively.
The method of claim 1, wherein the silver colloidal particles have a size of 1 nm to 100 nm, antimicrobial, antifungal and antiviral composition.
The method of claim 1, wherein the titanium dioxide nanoparticles have a size of 1 nm to 100 nm, antimicrobial, antifungal and antiviral composition.
A new species, comprising 0.0000005 to 10 parts by weight of silver colloidal particles, 0.0000001 to 15 parts by weight of titanium dioxide nanoparticles, 0.00001 to 6 parts by weight of at least one dispersion stabilizer, and 55 to 99 parts by weight of water Composition for influenza A (H1N1) virus killing.
The method of claim 12 wherein the dispersion stabilizer is lysolecithin, flavonoids, Tween 20, Tween 40, Tween 80, polyvinylpyrrolidone and polyvinyl alcohol (polyvinylalcohol) Swine influenza A (H1N1) virus killing composition, characterized in that any one or more selected from the group consisting of.
13. The composition for killing influenza A (H1N1) virus according to claim 12, wherein the silver colloidal particles have a size of 1 nm to 100 nm.
13. The novel influenza A (H1N1) virus killing composition according to claim 12, wherein the titanium dioxide nanoparticles have a size of 1 nm to 100 nm.
An anti-compound comprising 0.0000005 parts by weight to 10 parts by weight of silver colloidal particles, 0.0000001 parts by weight to 15 parts by weight of titanium dioxide nanoparticles, 0.00001 parts by weight to 6 parts by weight of at least one dispersion stabilizer, and 55 parts by weight to 99 parts by weight of water. A method of killing Swine Influenza A (H1N1) virus, comprising applying a Swine Influenza A (H1N1) virus composition.
The method of claim 16 wherein the dispersion stabilizer is lysolecithin, flavonoids, Tween 20, Tween 40, Tween 80, polyvinylpyrrolidone and polyvinyl alcohol (polyvinylalcohol) A method of killing a new influenza A (H1N1) virus, characterized in that at least one selected from the group consisting of.
17. The method of claim 16, wherein the silver colloidal particles are 1 nm to 100 nm in size.
17. The method of claim 16, wherein the titanium dioxide nanoparticles have a size of 1 nm to 100 nm.
A novel influenza A (H1N1) virus killing composition comprising 0.0000005 parts by weight to 10 parts by weight of silver colloidal particles, 0.0000001 parts by weight to 15 parts by weight of titanium dioxide nanoparticles, and 0.00001 parts by weight to 6 parts by weight of at least one dispersion stabilizer. Coated, Influenza A (H1N1) virus blocking filter.
21. The method of claim 20, wherein the dispersion stabilizer is lysolecithin, flavonoid, Tween 20, tween 40, tween 80, polyvinylpyrrolidone and polyvinylalcohol. New influenza A (H1N1) virus blocking filter, characterized in that any one or more selected from the group consisting of.
21. The novel influenza A (H1N1) virus blocking filter according to claim 20, wherein the silver colloid particles have a size of 1 nm to 100 nm.
21. The novel influenza A (H1N1) virus blocking filter according to claim 20, wherein the titanium dioxide nanoparticles have a size of 1 nm to 100 nm.
21. The new influenza A (H1N1) virus blocking filter according to claim 20, wherein the filter is made of polypropylene.
A new influenza A (H1N1) virus blocking mask comprising the filter of claim 20.