KR20160029969A - Virucidal composition containing natural products for avian inflenza virus - Google Patents

Abstract

Provided is an antiviral disinfectant composition which is eco-friendly and safe to domestic animals since a composite extract of medicinal herbal ingredients and a natural medicinal mixture are used. To this end, the composition comprises as active ingredients: an extract of natural medicinal herbal ingredients composed of Arimonia pilosa L., Cmmelina communis, and Sutellaria baicalensis; citric acid; and bamboo vinegar.

Description

[0001] The present invention relates to an avian influenza virus,

FIELD OF THE INVENTION The present invention relates to virucidal compositions, and more particularly to avian influenza virus antiviral compositions containing natural products.

The avian influenza (AI) is the most important disease causing great economic loss in the poultry and duck industries. The causative organism is the AI virus. At present, a vaccine for the prevention of AI has not been developed yet, and even if it is developed, there are many problems to exhibit a clear preventive effect due to a large variation due to the nature of the virus. In the absence of vaccine development, prevention of AI outbreaks is very important. In recent years, highly pathogenic AI has occurred in Korea, and the possibility of AI is increasing with climate warming.

Korean Patent Laid-Open Publication No. 2013-0083585 discloses a disinfectant composition which exhibits anti-cohesion, disinfection and sterilizing effects by controlling the combination ratio between organic and inorganic materials.

However, the conventional disinfectant composition is produced by chemical synthesis, which may adversely affect livestock and the environment. Therefore, it is inevitable to develop safer and environmentally friendly disinfectants for livestock.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a disinfectant for preventing avian influenza and preventing bacterial viral diseases in domestic animals using natural products. However, these problems are exemplary and do not limit the scope of the present invention.

According to one aspect of the present invention, there is provided a method for producing a natural herbal medicine extract, citric acid and bamboo vinegar comprising Arimonia pilosa L. , Cmmelina communis and Sutellaria baicalensis , ≪ RTI ID = 0.0 > antimicrobial < / RTI >

According to another aspect of the present invention, there is provided a pharmaceutical composition comprising a mixture of natural herbal medicine powder consisting of Agrimonia pilosa L. , Commelina communis and gold ( Scutellaria baicalensis ), water, C1 to C4 lower alcohol, An extraction step of extracting a complex extract by adding an aqueous solution of alcohol; And a step of blending and dissolving the extract powder with citric acid and bamboo shoots.

According to one embodiment of the present invention as described above, it is possible to realize an effective disinfection effect on environmentally-friendly avian influenza virus by blending citric acid, sunflower seeds, acacia var. Of course, the scope of the present invention is not limited by these effects.

Definition of Terms:

As used in this document, "bamboo vinegar" is a method of carbonizing bamboo in a closed space, cooling the gas (water vapor and smoke) produced during the process of producing charcoal, When left to stand for 3 to 6 months, it is divided into three layers, usually the middle layer. It contains 270 kinds of ingredients such as alcohols, phenols, carbonyls, neutral compounds and basic components in addition to acetic acid and other organic acids as main components of bamboo smoke extract. It has liver detoxification effect, liver function recovery effect, blood glucose lowering effect, hangover resolution and fatigue recovery Effect, antimicrobial and antiviral effect, and the like.

As used herein, " Agrimonia pilosa ledeb "is a perennial herbaceous perennial belonging to the Rosaceae family. The extracts of Seonghak-cho have been widely used as folk remedies, green juice, etc. and have been widely used as therapeutic agents for lung cancer, liver cancer, esophageal cancer, tumor, pain relief, branching, hematemesis, hematuria and uterine bleeding. In oriental medicine, it has strong convergence action and has been used as a medicinal and hemostatic agent. Agrimonin, agrimonolide, tormentic acid and ellagic acid, and luteolin-7-glucoside are contained in leaves and stems. The main components are agrimonin, agrimonolide, tormentic acid and ellagic acid. . Recent studies have reported antiviral, diabetic, vasodilatory, antimicrobial, antioxidant and anticancer activities.

As used in this document, " Commelina communis " belongs to the genus Chironomidaceae and is commonly known as a year-old grass common in roads and grasses. It is also called Chicken foxtail, Chicken underwoven, Chicken goby, and Starbuck. It has been used for antipyretic, detoxification, diuretic and has been known to be effective for diseases such as fever, jaundice, hepatitis, sore throat and diabetes caused by a cold. 1-carbomethoxy-β-carboline, norharman, and harman, which are alkaloid components, are reported to have blood pressure enhancing action, antimicrobial effect, antiviral effect and antioxidant effect.

As used herein, "Golden ( Scutellaria baicalensis )" is a perennial herb that belongs to the family Lamiaceae, and has long been used for the treatment of high fever, cough, constipation and jaundice. The roots include baicalein, baicalin, O-glucuronide, nobaicalein, sitosterol, and apigenin (also known as norepinephrine), wohonin, wogonin, wogonin 7-O-glucuronide, oroxylin A, apigenin) and chrysin (chrysin). Gold has been reported to have antibacterial, antiviral, liver protective, anti-cancer and anti-inflammatory effects.

As used herein, "citric acid" is a weak organic acid found predominantly in citrus fruits and is a colorless transparent powder. Citric acid is a natural preservative and is used as an acidifier to give acid or sour taste to food or beverage. Citric acid promotes the metabolism of carbohydrates in the body, has the effect of excretion of waste products in the body, and has been reported to have antibacterial and antifungal effects, antiviral effects, blood pressure lowering effects and fatigue recovery effects.

DETAILED DESCRIPTION OF THE INVENTION [

According to one aspect of the present invention, there is provided a method for producing a natural herbal medicine extract, citric acid and bamboo vinegar comprising Arimonia pilosa L. , Cmmelina communis and Sutellaria baicalensis , ≪ RTI ID = 0.0 > antimicrobial < / RTI >

In the composition, the natural herbal medicine may be composed of 20 parts by weight of Seokjak, 1 ~ 3 parts by weight of Angelica keiskei, 1 ~ 3 parts by weight of gold, And 2 to 6 parts by weight of a bamboo charcoal.

In the above composition, the virus may be a virus derived from poultry or livestock. The poultry-derived virus may be a newcastle disease virus or avian influenza virus (AI). The avian influenza virus may be avian influenza virus of the H5N1 or H9N2 type.

The livestock-derived virus may be a newcastle disease virus, a hog cholera virus, a H1N1 virus or FMDV virus.

In the composition, the bacterium may be Salmonella, Brucella, Escherichia coli, Pasteurella or Staphylococcus.

According to another aspect of the present invention, there is provided a pharmaceutical composition comprising a mixture of natural herbal medicine powder consisting of Agrimonia pilosa L. , Commelina communis and gold ( Scutellaria baicalensis ), water, C1 to C4 lower alcohol, An extraction step of extracting a complex extract by adding an aqueous solution of alcohol; And a step of blending and dissolving the extract powder with citric acid and bamboo shoots.

Hereinafter, the present invention will be described in more detail by way of examples. It should be understood, however, that the invention is not limited to the disclosed embodiments, but may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, Is provided to fully inform the user.

Example 1: Preparation of avian influenza virus viral composition

The present invention provides a sterilizing and virucidal composition using sunflower seeds, apricot herb, gold and citric acid, and bamboo shoots.

Specifically, the medicinal herbs such as sunflower seeds, ginger powder, and gold were pulverized and mixed at a ratio of 10: 1: 1, followed by shaking with methanol at room temperature for 24 hours. The supernatant of the natural product mixture was concentrated under reduced pressure, lyophilized extract powder and citric acid were mixed at a ratio of 1: 1 and dissolved in a bamboo liquid to a concentration of 20% to establish an AI virus antiviral composition. The composition and contents of the composition are shown in Table 1 below.

ingredient Content (g / L) Extract of natural materials 100 Citric acid 100 Bamboo flake Fit to total volume 1L

Example 2: Effectiveness test for composition disinfectant

To evaluate the efficacy of the AI virus antiviral composition prepared according to one embodiment of the present invention, a diluent containing a pathogen to be disinfected and a disinfectant diluent were mixed and then treated with the treatment.

2-1: Evaluation Criteria

According to the organic matter content of the object to be disinfected, the object to be disinfected is classified into a disinfection tank, floor, dirt, carcass, farm vehicle and transportation equipment, and the disinfection object with less organic material is classified into a housing space, . In addition, the effluent concentration was determined as the effective concentration of the dilution concentration at which the viral infectivity of the disinfectant treated group was inactivated by 10 ⁴ EID ₅₀ or more, compared with the viral infection activity of the pathogen control group. The results of the repeated test were converted into the logarithm of the common logarithm, and the result was regarded as the median of the results which were within the 20% error range.

2-2: Test virus

To evaluate the virucidal efficacy of the disinfectant composition prepared according to one embodiment of the present invention, the avian influenza virus H9N2 (MS96 strain) prevalent in the agriculture, forestry and livestock quarantine division was disclosed in the test. The virus strain was inoculated into the umbilical cord of 10-day-old AIV non-antibody SPF developmental cattle, subcultured, and tested at the time when the vital capacity of the proliferated virus was maximal (virus titer of 10 ⁷ EID ₅₀ / mL or more).

2-3: Dilution and disinfection of disinfectants

Dilution of disinfectant prepared according to one embodiment of the present invention was diluted with water and organic diluent (containing 5% fetal bovine serum) in accordance with the target treatment, and diluted with various dilutions according to the treatments. The dilution factors are shown in Table 2 below.

Condition Dilution factor Treatment 1 1 / 1,200, 1 / 1,350, 1 / 1,500, 1 / 1,650, 1 / 1,800, Treatment 2 1/800, 1/900, 1 / 1,000, 1 / 1,100, 1 / 1,200, 1 / 1,300

The disinfectant was prepared by mixing 1.0 mL of the uremic lavage fluid containing the virus into the hard water and organic diluent (5% fetal bovine serum) in a ratio of 19 mL to 2.5 mL each at 1 minute intervals, And mixed in a test tube. The mixed solution was reacted at 4 ° C for exactly 30 minutes and mixed every 10 minutes during the reaction. Treatment water 3 was replaced with hard water in place of disinfectant as a control. The hard water was prepared by dissolving 0.305 g of CaCl ₂ and 0.139 g of MgCl ₂ .6H ₂ O in 1 liter of distilled water, sterilized by high pressure and stored at 4 ° C. The organic dilution of the composition disinfectant was dissolved in sterilized hard water to contain 5% fetal bovine serum. After the reaction of the composition disinfectant was terminated, one part of phosphate buffered saline (pH 7.2) containing 10% fetal bovine serum immobilized at 1 minute of the composition disinfectant solution was neutralized at 37 ° C to neutralize the effect of the composition disinfectant immediately. In the 'Neutralization Reaction' stage, the titer of the pathogen was found to be 2 × 10 ⁵ TCID ₅₀ or more per ml. In the toxic control group of 4 treatments, the toxicity of the disinfectant Respectively. The treatments for the efficacy test evaluation of the disinfectants are shown in Table 3 below.

Treatment Water Organic matter disinfectant Remarks 1 (organic matter low) + - + Hard water condition 2 (organic matter) + + + Organic / light water conditions 3 (Pathogen contrast) + - - Control of treatments 1 and 2 4 (Toxic contrast) + - + Control of treatment 3

2-4: Determination of Virus Proliferation

Stock solution with a phosphate buffered saline to the neutralized liquid, 10 ^-1, 10 ^-2, 10 ^-3, 10 ^-4, 10 ^-5 and 10 ^-6 dilution of 0.2 ml after a 5-fold allowance 10 days balyukran diluted with , And cultured in an incubator at 37 캜 for 5 days. After that, the dogs were sacrificed every day and the dead cells within 24 hours after vaccination were regarded as accidents and excluded from the test results. The lumenal fluid was collected from all the inoculated dogs, and the infection was judged by the plate hemocyte aggregation reaction using the same amount of chicken blood (1%). The content of virus was calculated by the Kaerber method of Equation 1 below.

(L ₁ , the logarithm of the lowest dilution factor examined; L, the logarithm of the interval between dilution factors; S, the sum of the death rates at each dilution)

Example 3: Viral viral effect of disinfectant composition

The disinfectant composition prepared according to one embodiment of the present invention was diluted according to various dilution waters and treated in the experimental conditions to evaluate the disinfecting effect. The avian influenza virus was tested in triplicate for a dilution factor of 10 ⁴ reduction of pathogen in comparison with the control, and the median was set as the effective dilution factor.

3-1: First test result

As a result of evaluating the efficacy of the water-diluted solution containing the pathogen to be disinfected and the diluted organic solution with the same amount of the composition disinfectant diluent at 4 ° C for 30 minutes, the log reduction value was 4.0 at 1 / 1,500 dilution And in treatment 2, it was 4.1 in 1 / 1,000 dilution. The results of the above primary test are shown in Table 4 below.

Treatment
Disinfectant dilution
Neutral liquid dilution drainage (positive water / total inoculated water)
EID ₅₀
Log
Decrease value
10 ^-1 10 ^-2 10 ^-3 10 ^-4 10 ^-5 10 ^-6 Treatment 1




1 / 1,200 0/5 0/5 0/5 0/5 0/5 0/5 0.5 ≥5.8 1 / 1,350 2/5 1/5 0/5 0/5 0/5 0/5 1.1 4.3 1 / 1,500 3/5 2/5 0/5 0/5 0/5 0/5 1.5 4.0 1 / 1,650 4/5 3/5 2/5 1/5 0/5 0/5 2.5 3.4 1 / 1,800 5/5 4/5 3/5 2/5 1/5 0/5 3.5 2.7 1 / 1,950 5/5 5/5 5/5 3/5 2/5 1/5 4.7 1.8 Treatment 2




1/800 0/5 0/5 0/5 0/5 0/5 0/5 0.5 ≥5.8 1/900 1/5 0/5 0/5 0/5 0/5 0/5 0.7 5.1 1 / 1,000 3/5 1/5 0/5 0/5 0/5 0/5 1.3 4.1 1 / 1,100 3/5 2/5 1/5 0/5 0/5 0/5 2.1 3.5 1 / 1,200 5/5 5/5 4/5 2/5 1/5 0/5 3.9 2.4 1 / 1,300 5/5 5/5 5/5 3/5 2/5 1/5 4.7 1.8 Treatment 3
(Pathogen contrast) 5/5 5/5 5/5 5/5 5/5 4/5 6.3 Treatment 4
(Toxic contrast) 0/5 0/5 0/5 0/5 0/5 0/5

3-2: Second test result

In the second test, the log reduction value of 1 / 1,350 dilution drainage conditions at treatment 1 was found to be 4.1, and the 1/900 dilution drainage condition at treatment 2 was found to be 4.3. The results of the secondary test are shown in Table 5 below.

Treatment
Disinfectant dilution
Neutral liquid dilution drainage (positive water / total inoculated water)
EID ₅₀
Log
Decrease value
10 ^-1 10 ^-2 10 ^-3 10 ^-4 10 ^-5 10 ^-6 Treatment 1




1 / 1,200 0/5 0/5 0/5 0/5 0/5 0/5 0.5 ≥5.8 1 / 1,350 3/5 1/5 0/5 0/5 0/5 0/5 1.3 4.1 1 / 1,500 3/5 2/5 1/5 0/5 0/5 0/5 1.7 3.8 1 / 1,650 5/5 4/5 2/5 1/5 0/5 0/5 2.9 3.2 1 / 1,800 5/5 4/5 3/5 1/5 0/5 0/5 3.1 3.1 1 / 1,950 5/5 5/5 4/5 2/5 0/5 0/5 3.7 2.5 Treatment 2




1/800 0/5 0/5 0/5 0/5 0/5 0/5 0.5 ≥5.8 1/900 2/5 1/5 0/5 0/5 0/5 0/5 1.1 4.3 1 / 1,000 3/5 1/5 0/5 0/5 0/5 0/5 1.7 3.8 1 / 1,100 4/5 2/5 1/5 0/5 0/5 0/5 1.9 3.6 1 / 1,200 5/5 5/5 3/5 2/5 1/5 0/5 3.7 2.5 1 / 1,300 5/5 5/5 5/5 4/5 2/5 1/5 4.9 1.5 Treatment 3
(Pathogen contrast) 5/5 5/5 5/5 5/5 5/5 4/5 6.3 Treatment 4
(Toxic contrast) 0/5 0/5 0/5 0/5 0/5 0/5

3-3: Third test result

In the last tertiary test, the log reduction value of 1 / 1,500 dilution drainage condition was observed to be 4.3 in treatment 1, and the dilution drainage condition of 1 / 1,000 was 4.3 in treatment 2. The results of the third test are shown in Table 6 below.

Treatment
Disinfectant dilution
Neutral liquid dilution drainage (positive water / total inoculated water)
EID ₅₀
Log
Decrease value
10 ^-1 10 ^-2 10 ^-3 10 ^-4 10 ^-5 10 ^-6 Treatment 1




1 / 1,200 0/5 0/5 0/5 0/5 0/5 0/5 0.5 ≥5.8 1 / 1,350 0/5 0/5 0/5 0/5 0/5 0/5 0.5 ≥5.8 1 / 1,500 2/5 1/5 0/5 0/5 0/5 0/5 1.1 4.3 1 / 1,650 4/5 2/5 0/5 0/5 0/5 0/5 1.7 3.8 1 / 1,800 5/5 5/5 2/5 1/5 1/5 0/5 3.3 2.9 1 / 1,950 5/5 5/5 4/5 3/5 1/5 0/5 4.1 2.0 Treatment 2




1/800 0/5 0/5 0/5 0/5 0/5 0/5 0.5 ≥5.8 1/900 0/5 0/5 0/5 0/5 0/5 0/5 0.5 ≥5.8 1 / 1,000 2/5 1/5 0/5 0/5 0/5 0/5 1.1 4.3 1 / 1,100 4/5 2/5 0/5 0/5 0/5 0/5 1.7 3.8 1 / 1,200 5/5 5/5 4/5 2/5 0/5 0/5 3.7 2.2 1 / 1,300 5/5 5/5 5/5 4/5 2/5 1/5 4.9 1.5 Treatment 3
(Pathogen contrast) 5/5 5/5 5/5 5/5 5/5 4/5 6.3 Treatment 4
(Toxic contrast) 0/5 0/5 0/5 0/5 0/5 0/5

3-4: Test result judgment

Avian influenza viruses are effective dilution multiple set the median of the dilution factor a compared to the control agents is confirmed 10 ⁴ reduction in repeat conducted tests for three cars to estimate the one embodiment the effect of a disinfectant composition prepared according to the present invention Respectively. As a result, the median of the dilution factor of treatment 1 (organic matter low) was 1 / 1,500, the median of the dilution factor of treatment 2 (organic matter high) was 1 / 1,000 and no toxicity was observed in treatment 4. The results of the disinfectant efficacy test according to the dilution drainage of the disinfectant composition are shown in Table 7 below.

Treatment Primary Secondary Third Moderate number  Treatment 1 (organic matter) 1 / 1,500 1 / 1,350 1 / 1,500 1 / 1,500  Treatment 2 (organic matter) 1 / 1,000 1/900 1 / 1,000 1 / 1,000  Treatment 3 (Pathogen contrast) + + +  Treatment 4 (Toxic contrast) - - -

+, Virus titer 10 ⁶ EID ₅₀ / ml or more was confirmed.

-, no toxicity to the head.

Based on the test results, the recommended dilution ratios were determined to maximize the virulence effect of avian influenza viresicles according to the constitutional composition of Akecho, Golden, Seokhyeok, Citric acid and Bamboo liquid. The disinfection objects were classified into disinfection objects with high organic matter such as hard water condition, organic material condition, house space such as house space and house floor, and the dilution ratio was varied according to each composition. The recommended dilution factor checks for each of the above component compositions are shown in Table 8 below.

Disinfection
object

Recommended dilution
Composition A + E B + E C + E A + B + E A + C + E B + C + E A + B + D A + C + D B + C + D A + B + C + E A + B + C + D A + B + C + D + E Hard water condition 1: 200 1: 200 1:20 1: 500 1: 200 1: 200 1: 800 1: 400 1: 400 1: 1,000 1: 1,200 1: 1,500 Organic matter condition 1: 100 1: 100 1:10 1: 250 1:80 1:80 1: 400 1: 150 1: 150 1: 600 1: 700 1: 1,000 Organic matter Housing space etc. 1: 200 1: 200 1:20 1: 500 1: 200 1: 200 1: 800 1: 400 1: 400 1: 1,000 1: 1,200 1: 1,500 Organic matter House floor etc. 1: 100 1: 100 1:10 1: 250 1:80 1:80 1: 400 1: 150 1: 150 1: 600 1: 700 1: 1,000

As a result of the above tests, the disinfectant composition prepared by mixing citric acid with citric acid, herbal extracts, and herb extracts mixed with gold, and dissolving citric acid in a bamboo shoot, was subjected to an efficacy test on avian influenza virus three times. As a result, It was confirmed that when the composition was diluted 1,500 times in the hard water condition (low organic matter) and 1,000 times in the organic condition (high organic matter), it was found to have an effect on avian influenza virus.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

Claims (9)

The present invention relates to an extract of natural herbal medicine consisting of Arimonia pilosa L. , Cmmelina communis and gold ( Sutellaria baicalensis ), citric acid and bamboo vinegar as active ingredients, Virus composition. The method according to claim 1,
Wherein the natural oriental medicinal herb comprises 20 parts by weight of sunflower seeds, 1 to 3 parts by weight of crustacea, and 1 to 3 parts by weight of gold. The method according to claim 1,
Wherein the composition comprises 0.5 to 2 parts by weight of citric acid and 6 to 10 parts by weight of a bamboo shoot to 1 part by weight of the extract of the natural herbal medicine. The method according to claim 1,
Wherein the virus is derived from poultry or livestock. 5. The method of claim 4,
Wherein the poultry-derived virus is a newcastle disease virus, or avian influenza virus (AI). 6. The method of claim 5,
Wherein said avian influenza virus is H5N1 or H9N2 type. 5. The method of claim 4,
Wherein the livestock-derived virus is Pseudorabies virus, hog cholera virus, H1N1 virus, or foot-and-mouth disease virus (FMDV). The method according to claim 1,
Wherein the fungus is Salmonella, Brucella, Escherichia coli, Pasteurella or Staphylococcus. A natural herbal medicine powder composed of Agrimonia pilosa L. , Commelina communis , and gold ( Scutellaria baicalensis ) is mixed and an aqueous solution of water, C1 to C4 lower alcohol or the lower alcohol is added thereto to prepare a complex extract An extracting step of extracting;
And mixing and dissolving the extract powder with citric acid and bamboo shoots.