KR20150049438A - Composition for viral disease prophylaxis and/or cure of animal using purified bee venom - Google Patents

Abstract

The present invention relates to a composition for preventing and treating a viral disease by using refined bee venom and, more specifically, to a composition for preventing and treating a viral disease including apamin, phospholipase A_2, and melittin which are components of refined bee venom as active ingredients. A method for producing a composition for preventing and/or treating an animal viral disease according to the present invention comprises: a first step of producing a bee venom solution by mixing a venom raw ingredient with distilled water; a second step of removing foreign materials including non-water-soluble flower powder and dust by filtering the produced bee venom solution through a filtering sheet; a third step of obtaining a refined bee venom solution by filtering the bee venom solution removed with the foreign materials; and a fourth step of producing refined bee venom by freezing and drying the refined bee venom solution, wherein the bee venom includes 2.0-3.0% of apamin, 11.5-13% of phospholipase A_2, and 50-75% of melittin as active ingredients.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing and / or treating viral diseases in animals using purified bee venom,

The present invention relates to a composition for the prevention and treatment of viral diseases using tablet bee venom. More particularly, the present invention relates to a composition for preventing and treating viral diseases, which comprises as an active ingredient, afamin, phospholipase A ₂ and melitin, To a composition capable of preventing and treating diseases.

Bee venom refers to the poison that bees have, and consists of more than 40 substances including protein enzymes, peptide amino acids and active amines, including melittin, apamin and MCD-peptides, which are the main ingredients.

Bee venom has been used for arthritis, rheumatism and skin diseases since it has characteristics of analgesic, anti-inflammatory and antibacterial activity. In addition, modern pituitary hormone secretion promotion and promotion of blood circulation is known as the efficacy of multiple sclerosis, lyme disease (lyme disease) and chronic fatigue syndrome (chronic fatigue syndrome) is used as an alternative treatment for the treatment.

A virus consisting of a nucleic acid and a protein may infect a host capable of propagating a virus having a virus-infecting activity to cause a pathological change in a viral disease through infection with an affinity-imparting tissue. Several types of viruses can cause similar symptoms.

Antiviral drugs can be used for the prevention and treatment of secondary infections of bacilli. Especially, when a cell is infected with a virus, , Inflammation, extracellular formation and inclusion formation, and may be the cause of tumors.

Influenza virus (Influenza virus) belongs to the genus Orthomix virus and influenza virus. It infects the mucous membranes of the upper airway and causes respiratory diseases. These viruses are very infectious, and they are caused by bronchopneumonia, acute infectious diseases infectious disease, etc., and have characteristics such as high exchange rate and low mortality rate.

Influenza viruses have a very long immunological memory of antigenic stimulation, proliferate within the organism and cause segmental exchange, and can be isolated from pigs, horses, and ducks as well as humans.

In addition, Vesicular Stomatitis Virus (VES) is a virus based on non-segmented negative strand RNA which is a pathogen of vesicular stomatitis and is classified as rhabdovirus. The nucleoside bark of this viral RNA is spiral-symmetric, and virions (virus particles) have an outer membrane, the structure of which is shell-like. There is one kind of virus-specific protein (G protein) on the virion surface, and there is virus-specific RNA-dependent RNA polymerase inside the particle.

In addition, the Newcastle Disease Virus is an avian hospital virus in Paramixoviridae and Paramixovirus. Its size is about 150 to 200 nm in diameter, and it is encapsulated and has RNA polymerase in its particles.

Chicken Newcastle disease is a contagious pathogen. It causes bronchitis, pneumonia, diarrhea, spasms and paralysis. It causes high death rate. It can cause lethal lesions in other birds other than chickens and cause conjunctivitis in humans.

In addition, herpes simplex virus (Herpes Simplex Virus) round viral particles in the envelope inside the diameter of about 100nm on the surface of the icosahedral capsid. The genome is a double-stranded DNA and its size is, for example, 152,260 bases in herpes simplex type 1, 172,282 bases in EB virus, and 229,400 bases in cyto- megalovirus.

Herpes viruses proliferate in the nucleus of the host and produce inclusion bodies that are positive for Feulgen reaction. When the virus is reduced, it becomes a negative foilogen reaction and changes into a Cowdry A type inclusion body of eosinophilic (eosinophilic) (NITA) virus group.

Bee venom has an antiviral efficacy, which not only enhances the defense against viruses by stimulating the immune system in vivo in animals, but also has the effect of removing the toxin of target shingles virus.

Purified Bee Venom (PBV) is a non-water soluble pollutant, dust, etc. through natural processes such as drying, receiving, filtration, sterilization and freeze drying in order to maintain the natural state of bee venom collected by bee venom collecting device It is known that the bee venom of bee venom is similar to that of bee venom.

For example, Korean Patent Registration No. 10-1070600 discloses a composition for prevention and treatment of degenerative brain diseases comprising bee venom purified product isolated from bee venom as an active ingredient by using bee venom or bee venom as described above Korean Patent No. 10-1165510 discloses a composition for treating skin diseases such as reducing the weight gain of lymph nodes involved in T cell activity and reducing dermal and epidermal thickness using bee venom as an active ingredient .

A problem to be solved by the present invention is to provide a composition for preventing and / or treating viral diseases in an animal using purified bee venom containing apamin, phospholipase A ₂ and melitin as its main components.

The composition for the prevention, treatment, prevention or treatment of viral diseases in animals using tablet bee venom according to the present invention is useful as a pharmaceutical composition for preventing and / or treating viral diseases in an animal, which comprises an agent selected from the group consisting of afamin, phospholipase A ₂ and melittin As an active ingredient.

The method for preparing a composition for preventing and / or treating a viral disease of an animal according to the present invention comprises a first step of producing a bee venom aqueous solution by mixing a bee venom raw material with distilled water, A second step of removing impurities including water soluble pollen and dust, a third step of filtering the bee venous aqueous solution from which the impurities have been removed with a filter membrane to obtain an aqueous solution of the bee keeping solution, and a third step of freeze- And the fourth step is characterized in that the tablet bee venous contains as an active ingredient 2.0 to 3.0% of apamin, 11.5 to 13% of phospholipase A ₂ , and 50 to 75% of melitin.

According to the composition for the prevention and / or treatment of viral diseases in animals using the tablet bee venom of the present invention, it is possible to provide a composition for preventing and / or treating viral diseases in an animal, comprising 2.0 to 3.0% of afamin, 11.5 to 13% of phospholipase A ₂ , 50 to 75% of melitin The use of purified bee venom as an active ingredient can prevent and treat diseases caused by viruses in animals.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a flow chart showing a method for preparing a composition for preventing and / or treating viral diseases in animals using tablet bee venom according to the present invention.
Fig. 2 is a diagram showing the components of the tablet beacon prepared by the embodiment of the present invention. Fig.
Fig. 3 is a diagram showing a preventive effect of the tablet beeing according to the present invention acting on influenza virus in macrophages. Fig.
FIG. 4 is a graph showing a preventive effect of tablet beeing according to the present invention on macrophage polio virus infection in macrophages. FIG.
FIG. 5 is a graph showing a preventive effect of tablet beeing according to the present invention on the vesicular stomatitis virus in epithelial cells. FIG.
Fig. 6 is a graph showing the preventive effect of the tablet beeing according to the present invention on the action of herpes virus in macrophages. Fig.
Fig. 7 is a graph showing the preventive effect of the tablet beeing according to the present invention on the herpes virus in epithelial cells. Fig.
Fig. 8 is a diagram showing a preventive effect of tablet beeing according to the present invention on macrophage to Newcastle virus. Fig.
9 is a view showing the effect of tablet beeing according to the present invention in inducing secretion of cytokines and interferons in macrophages.
FIG. 10 is a diagram showing the therapeutic effect of tablet beeing according to the present invention on macrophage colitis virus in macrophages. FIG.

Hereinafter, the present invention will be described in more detail. It is to be understood, however, that the present invention is not limited to the embodiments described below.

FIG. 1 is a flowchart showing a process of purifying the collected bee venom to produce a tablet bee venom.

According to the flow of FIG. 1, in the process of purifying bee venom collected from a bee to produce a purified bee venom, the bee venom raw material collected is mixed with distilled water to produce an aqueous solution (S100). The aqueous solution can be mixed and dissolved in a bee venom raw material collected from a bee and distilled water at a weight ratio of, for example, 1:10.

At this time, it is necessary to adjust the amount of distilled water to an adequate amount in order to sufficiently pass the active ingredient through the following filtration step and recover it. If the amount of distilled water is insufficient, the recovery rate is decreased. If the amount of distilled water is too large, the efficiency of the filter and final drying of the next process is lowered.

Next, the resulting aqueous solution is filtered through a filter paper to remove foreign matter including water-insoluble pollen and dust (S200). The filter paper is subjected to reduced pressure filtration using, for example, a filter paper of 3 mu m.

Bee venom is composed of water-soluble active ingredients such as protein enzymes, peptides, amino acids and active amines, and foreign substances such as water-insoluble pollen and dust are removed in the above-mentioned filtration process.

Next, the bee venom aqueous solution from which the foreign substances have been removed is filtered with a filter membrane to obtain a purified beacon aqueous solution (S300).

Filtration using the filter membrane can proceed to two steps. First, pressurization filtration is performed, for example, by using a 0.45 탆 filter membrane to remove fine foreign substances and the like. Next, a 0.2 μm filter membrane is used to perform pressure filtration to remove microorganisms, bacteria, and the like.

Thereafter, an aqueous solution from which foreign substances have been removed is freeze-dried to produce tablets bee poison (S400). The tablet bee venom obtained above contains 2.0 to 3.0% of acamin as an active ingredient, 11.5 to 13% of phospholipase A ₂ , and 50 to 75% of melitin.

Evaluation and analysis of tablets bee venom

1) Analysis of components of tablet bee venom

Each sample was accurately prepared at a concentration of 1.0 mg / mL for analysis of quantitative and qualitative analysis of the active ingredient of tablets prepared by the method according to the present invention, and an HPLC (high performance liquid chromatography) apparatus and a column for peptide analysis (150 X 4.6 mm 4.0 ㎛, 90Å, phenomenex ^®) which was analyzed by HPLC in water (0.2% TFA (trifluoroacetic acid) in water) and acetonitrile (0.22% TFA in ACN ( acetonitrile)) for use.

As a result, FIG. 2 shows that the peak of apamin and melitin is concentrated at 10.47 min and 22.25 min, and the peak of phospholipase A ₂ is 16.68 min It can be confirmed that it has been leaked to. The calibration curve of the standard and the area of each component were calculated and the content of each component was calculated through the following Equation 1 and shown in Table 1.

[Formula 1]

[Table 1]

2) Safety evaluation of tablet bee venom

The safety evaluation of tablets prepared by the method of the present invention was evaluated and registered (FDA-DMF 27321) through the request of a stability evaluation institution designated by the US FDA-DMF (US Food and Drug Administration). Specific experiments were performed at the concentrations of 0.1 mg / ml and 0.2 mg / ml of purified bee venom according to the FDA Good Laboratory Practice (GLP) standard as follows.

L929 cells are cultured and stored in MEM (Minimum Essential Medium (Eagles)) and EBS (Earles Balanced Salts) media, and nutrients are given to cells by mixing fetal bovine serum and antibiotic-antifungal solution. The sterile tissue culture grade of 60mm in Petri plates (cell culture) at a density of after trypsin (Trypsin) treatment on confluent (confluent) cell cultures, passages per plate to the culture (subculture) of about 1 x 10 ⁶ cell Cells are cultured. All plates are incubated for 24 hours or more in a humidified environment of 37 ± 1 ° C containing 5 ± 1% CO ₂ to obtain a confluent monolayer of about 80% in the experiment.

After incubation, the morphology of the subconfluency of cultured cells is confirmed using a microscope. Cultured cells containing normal and healthy cells were used in the experiments in cultured cells that were approximately 80% confluency (80% confluency) of the plate.

The medium was carefully removed from each cell monolayer and replaced with 1: 1 mixed 2% w / v purified agar (agar medium) and 2x Complete MEM 7ml. Stack the agar at room temperature for at least 15 minutes and stain the cell monolayer with 2-5 ml of 0.01% neutral red stain before beginning the experiment. Remove the staining solution after staining for at least 15 minutes.

Place sterilized filter paper (~ 2.1 cm ² ), which contains approximately 0.2 ml of the appropriate concentration of the test substance, on the agar surface to enclose approximately 10% of the cell agar layer surface.

Prepare the filter paper with filter paper soaked in sterile distilled water for injection. Standard positive or negative control material is prepared by sterilizing latex rubber and USP HDPE standard samples to 2.1 cm ² size and placed on agar surface.

All tests are repeated 3 times and 3 plates are prepared as blank (control group). All plates are incubated for 24 to 28 hours in a humidified environment of 37 ± 1 ° C with 5 ± 1% CO ₂ .

The test results are shown in Table 2. The evaluation criteria are appropriate for cell experiments when the negative control value is 0 and the positive control value is 3, even though the value is low.

When the value of the sample of bee venom is not more than 2, the condition as the test substance can be satisfied. When a value of 2 or higher appears, it is considered to be toxic.

[Table 2]

Efficacy test of purified bee venom for virus

The efficacy of the bee venom of the present invention in the prevention and treatment of viral diseases was tested in influenza virus, vesicular stomatitis virus, Newcastle virus and herpes virus in macrophages and epithelial cells of animals.

The efficacy of the tablet bee venom was compared with that of the virus-gfp by varying the concentration of the tablets bee venom.

1) Prevention of virus

The cells were pretreated with 12-hour pre-incubated cells (macrophage: Raw 264.7; epithelial cell: Human Kidney Cell Line 293T) and then cultured for another 12 hours. Viruses are infected with influenza virus (PR8-FGP), vesicular stomatitis virus (VSV-GFP), Newcastle virus (NDV-GFP) and herpes viruses (HSV-GFP) that express Green Fluorescence Protein . The infected cells are cultured again for 12 hours to measure the expression of GFP due to virus multiplication. The lower the expression of GFP, the higher the effect of virus prevention.

As a result, virus infection and proliferation rate were compared according to the amount of tablets beeing, as shown in FIGS. 3 to 8, to determine the effect. Each figure shows the GFP expression of the virus when administered in different amounts of the tablet bee venom in macrophages or epithelial cells compared to the case of not receiving the tablets bee venom. A common feature of each figure is that the expression of GFP is markedly reduced when a larger amount of purified bee venom is administered to the virus. This means that the purified bee venom of the present invention remarkably exhibits the reduction and inhibitory effect on the infection and proliferation rate of the virus in the experiment on the preventive effect of the virus infection.

Figure 3 shows the effect of tablet bee venom to prevent influenza virus infection in macrophages.

FIG. 4 and FIG. 5 show the effect of tablet bee venom to prevent infection of virus-like stomatitis virus in macrophages and epithelial cells, respectively, and it can be confirmed that not only in macrophages but also in epithelial cells.

FIGS. 6 and 7 show the effect of tablet beeing to prevent herpes virus infection in macrophages and epithelial cells, respectively.

Figure 8 shows the effect of tablet bee venom to prevent Newcastle virus infection in macrophages.

The fact that the purified bee venom acts on the immune cells and shows the antiviral effect against various viruses indicates that a specific substance such as apamin, phospholipase A ₂ and melitin among the constituents of the tablets bee stimulates the immune cells, Which is the result of induction to inhibit proliferation.

9 shows the effect of tablet bee venom inducing the secretion of cytokines and interferons from macrophages. It was confirmed that secretion of inflammatory cytokines and interferons was induced with time, The secretion of cytokines such as TNF-α, IL-6 and interferon-β was also confirmed. As shown in the graph, the amount of cytokine and interferon increased as the amount of purified bee venom was increased.

In the case of epithelial cells, the mechanism of response to external stimuli was weaker than that of immune cells. This means that specific substances such as apamin, phospholipase A ₂ and melitin among the components of tablet bee venom stimulate not only immune cells but also epithelial cells to inhibit the proliferation of viruses.

2) Therapeutic effect of viral diseases

The therapeutic effect of the purified bee venom prepared by the method according to the present invention is infected with a mixture of tablets bee venom (VSV-GFP) and virus bee venom (VSV-GFP), which has been previously reacted with macrophage Raw 264.7 for 20 minutes. Then, the infected cells were cultured for 22 to 24 hours, and the expression of GFP due to virus multiplication was measured.

As shown in FIG. 10, it was confirmed that infection and proliferation rate of VSV were remarkably decreased as the amount of tablets bee venom to be administered was increased.

Claims (3)

A composition for preventing, treating, or preventing and treating viral diseases in animals using a tablet bee venous containing apamin, phospholipase A ₂ and melitin as an active ingredient.
The method according to claim 1,
The prevention, treatment, or prevention of viral diseases in animals using the tablet bee venom is characterized in that the above-mentioned apamin contains 2.0 to 3.0%, the phospholipase A ₂ contains 11.5 to 13% and the melittin contains 50 to 75%. ≪ / RTI >
A first step of mixing the bee venom raw material with distilled water to produce an aqueous bee venom solution;
A second step of filtering the generated aqueous solution of bee venom with a filter paper to remove foreign matter including water-insoluble pollen and dust;
A third step of filtering the bee venous aqueous solution from which foreign substances have been removed with a filter membrane to obtain a purified bee keeping aqueous solution; And
And a fourth step of lyophilizing the purified bee keeping solution to prepare a tablet bee;
Wherein said tablet bee venom is comprised of 2.0 to 3.0% of apamin, 11.5 to 13% of phospholipase A ₂ , and 50 to 75% of melitin as active ingredients. , ≪ / RTI >

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