KR102449133B1 - Antiviral composition and preparation method thereof - Google Patents

Abstract

The present invention relates to an antiviral composition comprising a lysosome isolated from a eukaryotic cell. Furthermore, the present invention provides a method for preparing lysosome composition having optimal antiviral activity. The composition comprising a lysosome according to the present invention can be used not only for spaces requiring virus removal such as masks, hands, garments, instruments, articles, and the like, but also for any place where antiviral activity is required during storage or use of pesticides, pharmaceuticals, food, cosmetics, animal drugs, feed, drinking water, and the like.

Description

Antiviral composition and its preparation method {Antiviral composition and preparation method thereof}

The present invention relates to an antiviral composition comprising a lysosome of a cell and a method for preparing the same.

Recently, as viral infectious diseases are prevalent around the world, research on materials that reduce the possibility of virus spread by killing or inactivating viruses floating in the air as antiviral substances and daily life prevention to prevent the spread of infectious viral diseases are actively being conducted. is in progress

Viruses are intermediate beings between living and non-living things in the cells of other living things. Representative viral infectious diseases include influenza, human immunodeficiency, coronavirus infection, Zika virus, and MERS, which affect variously from mild to severe.

According to the World Health Organization, not only COVID-19, which is currently prevalent, but also viral infectious diseases that are transmitted from person to person are known to be spread through airborne spread such as coughing or sneezing. have. Existing items used for quarantine are being quarantined using alcohol and hypochlorite-based substances, but alternative countermeasures are not met due to side effects due to the misuse and abuse of chemical components and side effects due to the toxicity of the quarantine products and disinfection products using them. Demand is increasing.

Registered Patent No. 2124592 discloses a hand sanitizer composition having antiviral activity comprising a bracken ethanol extract and a maca extract as active ingredients.

Registered Patent No. 2110663 discloses a disinfectant for swine diarrhea virus and respiratory virus containing an ethanol extract of king mushroom.

However, development of various materials is required for the smooth supply of antiviral agents.

As the pandemic of viral infectious diseases, including COVID-19, has emerged as a major problem and the demand for virus prevention and disinfection products is rapidly increasing, the present application is an environmental and human-friendly antiviral material, a composition comprising the same, and manufacturing the composition An object of the present invention is to provide a method and use of the composition.

The problem to be solved by the present invention is not limited to such a verbal description, and includes everything that a person skilled in the art can infer through the present invention.

As a result of intensive research to achieve the above object, the present inventors have found that lysosomes present in cells, particularly lysosomes isolated from yeast, have excellent antiviral activity.

Accordingly, the present application provides, in one aspect, an antiviral composition comprising a lysosome isolated from a eukaryotic cell.

In addition, the present application provides an antiviral composition comprising, in one aspect, lysosomes isolated from yeast.

In one aspect, the present application provides a virus disinfectant composition comprising a lysosome isolated from a eukaryotic cell.

In one aspect, the present application provides a disinfectant composition for an enveloped virus comprising a lysosome isolated from a eukaryotic cell.

In one aspect, the present application provides a disinfectant composition for porcine reproductive and respiratory syndrome virus comprising lysosomes isolated from eukaryotic cells.

The present application, in one aspect, provides a method for preparing an antiviral composition comprising a lysosome.

In one aspect, the present application provides a method of removing a virus using a lysosome isolated from a eukaryotic cell.

In one aspect, the present application provides a pharmaceutical composition for preventing or treating a viral infection comprising a lysosome isolated from a eukaryotic cell.

The lysosome composition having antiviral activity provided in the present invention is a cell-derived eco-friendly material, characterized by high antiviral activity and human friendliness. It can also be used for disinfection and cleaning of instruments and articles, thereby contributing to the prevention of the spread of viral diseases.

In addition, the lysosome composition having antiviral activity provided in the present invention can be utilized where antiviral activity is required during storage or use of eco-friendly pesticides, pharmaceuticals, food, cosmetics, animal drugs and feed additives.

Furthermore, the lysosome composition according to the present invention can be used for prevention and treatment of diseases caused by viral infection.

The effects of the present invention are not limited to such literature descriptions, and include all those that those skilled in the art can infer through the present invention.

1 is a lysate test result when Pseudomonas pi 6 virus was treated with a lysosome composition separated in Example 1 and stored at 4 ^o C for 1 week.
2 is a lysate test result by the lysosome composition for Pseudomonas pi 6 virus according to the storage period of the lysosome composition separated in Example 1 and stored at 4 ^o C (0 days, 7 days, 14 days, respectively) to be.
Figure 3 is the result of observing the effect of the lysosome composition according to the present invention on the cellular lesions of monkey kidney-derived cells MARC145 cells caused by pig reproductive and respiratory syndrome virus.

The present invention relates to the use of antiviral activity of lysosomes isolated from eukaryotic cells.

Lysosomes are organelles responsible for digestion of substances such as proteins, lipids, and nucleic acids inside and outside eukaryotic cells. These lysosomes include about 60 types of hydrolytic enzymes necessary for material degradation, such as proteolytic enzymes and lipolytic enzymes. Representative enzymes include acid dephosphatase, ribonucleic acid hydrolase, gathepsin, β-glucloritase, and arylsulfatase.

Viruses can only multiply in host cells and contain single or double strands of genetic material. Infection begins through replication and proliferation of genetic material in host cells and causes disease. The genetic material inside the virus, which is the key to viral infection, is protected by two or three layers, depending on the virus species. The protective composition of the viral genetic material consists of lipids, proteins and nucleic acids.

The inventors of the present invention separate the lysosomes retaining enzyme activity from yeast using cell disruption and centrifugation, and Pseudomonas pi 6 virus used as a representative model of virus experiments using the lysosome composition according to the present invention (Pseudomonas) phi6) and porcine reproductive and respiratory syndrome virus (PRRSV) for Plaque assay, it was confirmed that the lysosome according to the present invention had antiviral activity (Table 1). , see FIGS. 1 and 3).

In addition, according to the period of refrigeration storage at 4 ^o C after separation from the lysosome composition according to the present invention, the virus killing ability increases to 44.82% on the 0th day, 99.23% on the 7th day, and 99.4% on the 14th day. was found (see Fig. 2). The present inventors confirmed the antiviral activity of the lysosome composition according to the present invention and completed the present invention, and based on the research results thereof, the following are provided.

In one aspect, the present invention provides an antiviral composition comprising a lysosome isolated from a eukaryotic cell. The eukaryotic cell may be selected from animal cells, plant cells, yeast, and the like, but is not limited thereto.

In one embodiment of the present invention, the present invention provides an antiviral composition comprising lysosomes isolated from yeast.

In addition, in one aspect, (a) centrifuging the cultured cells to recover the cells, (b) adding a breaking buffer and disrupting the cells, (c) centrifuging to obtain a supernatant Lysis isolated from eukaryotic cells, comprising the steps of (d) centrifuging the obtained supernatant to collect the lysosomes from the lower layer, and (e) refrigerated storage of the collected lysosomes at 4 ^o C. A method for preparing a body composition is provided.

In one embodiment of the present invention, in step (a), the cells may be recovered by centrifugation at about 3,000 rpm. In one embodiment, in the present invention, Tris-SO ₄ solution (0.01M Dithiothreitol, 0.1M Tris-SO4) is added to the recovered cells and resuspended, the cells are cultured and then left on ice for 5 minutes, The method may further include re-collecting the cells by centrifugation at 3,000 rpm.

In one embodiment of the present invention, the breaking buffer of step (b) may be 1X Protease inhibitor cocktail, 20mM Tris-HCl, 0.6M Sorbitol. Also, in an embodiment of the present invention, 2 to 10 ml of the breaking buffer may be added per cell weight (g), but is not limited thereto. In one embodiment of the present invention, step (b) may include disrupting cells by repeating vortexing and leaving on ice after adding a breaking buffer.

In one embodiment of the present invention, the step (c) may be centrifuged at 500xg to obtain a supernatant, but is not limited thereto.

In one embodiment of the present invention, the step (d) may be centrifuged at 20,000xg, but is not limited thereto.

In one aspect, the present invention provides an antiviral composition comprising a lysosome prepared according to the above method.

In one aspect, the present invention provides a method of removing a virus using the lysosome composition according to the present invention.

In one embodiment of the present invention, the subject using the lysosome composition according to the present invention is a mask, hand, clothes, appliance, article, space requiring virus removal, etc., as well as pesticides, pharmaceuticals, food, cosmetics, veterinary drugs, It can be used wherever antiviral activity is required during storage or use, such as feed and drinking water, but is not limited thereto.

In one aspect, the present invention provides a composition for a virus disinfectant comprising a lysosome according to the present invention.

In one aspect, the present application provides a disinfectant composition for an enveloped virus comprising a lysosome isolated from a eukaryotic cell. Encapsulated virus refers to a type of virus that has a film covering the outside of the capsid of the virus, and includes influenza virus, herpes virus, coronavirus, HIV, HBV, HCV, and the like.

In addition, in one aspect, the present invention provides a composition for disinfectant of porcine reproductive and respiratory syndrome virus.

In one aspect, the present invention provides a composition for a cleaning agent comprising a lysosome according to the present invention.

In one aspect, the present invention provides a virus disinfectant comprising a lysosome according to the present invention.

In addition, in one aspect, the present invention provides a cleaning agent comprising a lysosome according to the present invention.

As an embodiment of the present invention, the disinfectant or cleaning agent or antiviral composition comprising lysosomes according to the present invention may include a buffer for optimizing the activity of enzymes contained in the lysosomes. The buffer may be, for example, pH 5.0 to 7.0, but is not limited thereto. The buffer may be, for example, a phosphate buffer, but is not limited thereto.

In one aspect of the present invention, the isolated lysosome composition according to the present invention can be used for the prevention or treatment of infectious diseases caused by viruses. The pharmaceutical composition comprising the lysosome according to the present invention may be administered to a human, mammal, livestock or poultry infected with a virus, but is not limited thereto.

The term "livestock" is a concept that refers to useful animals that have been domesticated and improved by humans and live together with humans, and includes, for example, pigs, cattle, chickens, horses, ducks or dogs, but is limited thereto. it's not going to be

The term "poultry" is a concept that collectively refers to animals belonging to birds among livestock, and includes, for example, chickens, ducks, pheasants, quails, ostriches, geese or turkeys, but is not limited thereto.

The pharmaceutical composition comprising the lysosome of the present invention may be administered to an animal in the form of a pharmaceutical formulation, or may be administered by mixing it with livestock feed or drinking water and feeding it.

The pharmaceutical composition according to the present invention may be administered through a variety of oral or parenteral routes as long as the administration route of the composition can reach the target tissue, and specifically, oral, rectal, topical, intravenous, intraperitoneal, intramuscular , intraarterially, transdermally, intranasally, via inhalation, and the like.

It is apparent to those skilled in the art that a suitable total daily amount of the lysosomes administered in the method of the present invention can be determined by a treating physician within the scope of sound medical judgment. A specific therapeutically effective amount for a specific individual may be determined together with the type and degree of response to be achieved, the patient's age, weight, general health, sex and diet, administration time, administration route and secretion rate of the composition, treatment period, and specific composition. It is preferable to apply differently depending on various factors including drugs used or concurrently used and similar factors well known in the medical field.

Terms not defined otherwise herein have the meanings commonly used in the technical field to which the present invention pertains.

Hereinafter, a method for preparing a lysosome composition from a cell according to a specific embodiment of the present invention and the antiviral effect of the composition will be described in more detail. However, this is presented as an example of the invention, thereby not limiting the scope of the invention, it is apparent to those skilled in the art that various modifications to the embodiment are possible within the scope of the invention. Matters that are not different from the prior art and are not necessary to understand the technical spirit of the present invention are excluded from the description.

<Example 1> Preparation of lysosome composition containing enzyme complex

Lysosomes were isolated from yeast cells through cell disruption and centrifugation to prepare a lysosome composition containing the enzyme complex.

More specifically, the yeast Saccharomyces cerevisiae was cultured in YPD medium (1% yeast extract, 2% peptone, 2% D-glucose) at 30 ° C. 180 rpm for 24 hours. The yeast cell culture medium was centrifuged at 3,000 rpm for 5 minutes to recover the cells. 5ml Tris _- SO4 solution (0.01M Dithiothreitol, 0.1M Tris-SO4) was added to the recovered cells per cell weight (g) and resuspended, and the cells were cultured at 30 °C and 90 rpm for 24 hours and then placed on ice for 5 hours. It was left for a minute. Thereafter, the cells were recovered by centrifugation at 3,000 rpm for 5 minutes, and then 4ml of breaking buffer (1X Protease inhibitor cocktail, 20mM Tris-HCl, 0.6M Sorbitol) was added per cell weight (g). Then, vortexing for 1 minute and leaving on ice for 1 minute were repeated 10 times to disrupt the cells. After cell disruption, centrifuge at 500xg for 5 minutes, transfer the supernatant to a microtube, centrifuge at 4℃, 20,000xg for 30 minutes, discard the supernatant of the separated solution, collect the lower lysosomes, and add sterile tertiary distilled water. , refrigerated at 4 ^o C.

<Example 2> Confirmation of virus inhibition activity - gyunban test

In order to confirm the virus inhibitory activity of the lysosome composition made in Example 1, Pseudomonas phi6 virus and porcine reproductive and respiratory syndrome virus; PRRSV used as a representative model of virus experiments. ) for plaque assay (Plaque assay) was performed.

Pseudomonas pi 6 virus (hereinafter also referred to as pi 6 virus) and its host cell, Pseudomonas syringae, used as representative specimens of enveloped viruses were cultured and inoculated. Infection was confirmed by plaque formation. Pseudomonas pi 6 and Pseudomonas syringe were purchased from DSMZ (Pseudomonas phage phi6::DSM 21518, Pseudomonas syringae::DSM 21482), and PRRSV was purchased from the laboratory of Professor Hyekwon Kim of Chungbuk National University. Cultivation of Pseudomonas siringe was cultured in Tryptic Soy Broth (1.7% Pancreatic digest of Casein, 0.3% Papaic digest of Soybean, 0.25% Dextrose, 0.5% Sodium Chloride, 0.25% Dipotassium Phosphate) at 26 ° C. 180rpm for 24 hours. Concentration was run at OD (600 nm): 0.8. The concentration of the lysosomes treated with the virus was based on the protein concentration, and the lysosomes having a protein concentration of 10ug/ul were dissolved in sterile distilled water at 1:1 to treat a volume ratio of 10%. The Tryptic Soy Broth used for the lysate test used the same composition used for culturing Pseudomonas siringe, and the agar concentration of the bottom agar plate was 7% and the agar concentration of the top agar was 3.5%.

In the group treated with the lysosomes separated in Example 1 and stored at 4 ^o C for 1 week, the virus killing rate was 99.23% for pi 16 virus and 99.80% for the porcine reproductive and respiratory syndrome virus, according to the present invention. It was confirmed that the lysosome has antiviral activity (see Table 1 and FIG. 1).

<Example 3> Antiviral activity according to storage period after lysosome separation

The antiviral activity according to the period of storage of the lysosome composition isolated according to Example 1 at 4 ^o C was investigated against pi 16 virus according to Example 2. When the lysosome composition having a storage period of 0 days, 7 days and 14 days was used, the virus killing rates were 44.82%, 99.23% and 99.4%, respectively (see FIG. 2 ).

<Example 4> Confirmation of virus inhibitory activity - Cell lesion test

In order to confirm the virus inhibitory activity of the lysosome composition prepared in Example 1, a cell lesion test for Porcine reproductive and respiratory syndrome virus (PRRSV) was performed.

MARC145 cell, a monkey kidney-derived cell (received from the laboratory of Professor Hye-Kwon Kim, Chungbuk National University) was inoculated with 5Х104 cells per well in 96-well plating. Thereafter, PRRSV (1.00Х105 TCID50/mL) was treated with a lysosome composition (100ug/mL), and then the host cell, MARC145 cell, was infected and the cytopathic effect (CPE) was confirmed through a microscope.

In the experimental group treated with the lysosomes, it was confirmed that the adhered cells did not fall off and there was no cell lesion, and it was confirmed that the lysosomes according to the present invention had a virus killing effect and an antiviral effect (FIG. 2).

Although the present invention has been described above, the present invention is not limited by the disclosed embodiments and the accompanying drawings, and various modifications may be made by those skilled in the art within the scope without departing from the technical spirit of the present invention. In addition, the technical ideas described in the embodiments of the present invention may be implemented independently, or two or more may be implemented in combination with each other.

Claims (10)

delete delete delete (a) centrifuging the cultured yeast cells to recover the cells,
(b) adding a breaking buffer and disrupting the cells;
(c) centrifuging at 3000xg to obtain a supernatant;
(d) centrifuging the obtained supernatant at 20,000xg to collect the lysosomes from the lower layer, and
A method for producing lysosomes isolated from yeast cells, comprising the step of refrigerated storage of the collected lysosomes at 4 ^o C for at least 1 week.
An antiviral composition comprising a lysosome prepared according to the method of claim 4.
A method for removing viruses using the composition according to claim 5 .
A composition for a virus disinfectant comprising a lysosome prepared according to the method of claim 4.
The composition according to claim 7, for disinfecting porcine reproductive and respiratory syndrome virus.
A composition for a cleaning agent comprising a lysosome prepared according to the method of claim 4.
A pharmaceutical composition for preventing or treating a viral infection comprising a lysosome prepared according to the method of claim 4 .

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