WO2022131603A1 - Antiviral composition containing geranium herb extract as active ingredient - Google Patents

Abstract

The present invention relates to an anti-coronavirus use of an antiviral composition containing a Geranium herb extract as an active ingredient. Specifically, the present invention relates to the development of an antiviral agent for preventing or treating viral diseases since a Geranium herb extract of the present invention exhibits an inhibitory effect on an infection caused by coronavirus such as SARS-coronavirus-2 (SARS-CoV-2).

Description

Antiviral composition comprising a sagebrush extract as an active ingredient

The present invention relates to an antiviral composition comprising an extract of chrysanthemum as an active ingredient, and more particularly, to a use for preventing, improving or treating SARS-CoV-2 infection comprising the extract as an active ingredient.

Coronavirus was first discovered in chickens in 1937, followed by animals such as dogs, pigs, and birds, and then in humans in 1965. During a total solar eclipse, when the sun's photosphere is obscured by the moon, it is given this name because its appearance is similar to the corona phenomenon, which is a phenomenon that glows white around the moon.

Coronaviruses are known to mainly cause pneumonia and enteritis in humans and animals, and are also known to occasionally cause nervous system infections and hepatitis. Coronavirus belongs to the coronaviridae and is a positive sense RNA virus with a spherical outer membrane and a size of about 100-120 nm (Master, 2006). Coronavirus consists of a total of five structural proteins: the outermost spike protein (S), hemagglutinin-esterase (HE) protein, transmembrane (M) protein, small membrane (E) protein, and nucleocapsid (N) protein. There is (Lai and Homes, 2001. Fields Virology). Among them, the spike protein acts as a ligand that binds to the cell receptor (ACE2) and induces fusion between the host cell and the virus, and is known as the most mutated protein.

So far, coronavirus has been recognized as a pathogen that rarely infects humans and mainly infects animals such as dogs, pigs, and cattle. It is one of several viruses that cause respiratory symptoms even when infecting humans, and only causes a simple cold or intestinal disease such as diarrhea, which is not very dangerous for children. However, as the causative agent of severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS), which caused more than hundreds of deaths and thousands of patients worldwide, are known to be novel (variant) coronaviruses, gradually began to attract attention.

SARS is an infectious disease that originated in Guangdong Province in southern China in November 2002 and spread to the world through Hong Kong. The path of propagation is not yet fully elucidated, but it is speculated that it is propagated by fine particles of solid or liquid floating in the atmosphere. The SARS epidemic lasted until July 2003, and in about 7 months, a total of 8,096 infections were reported in 32 countries, and 774 of them were known to have died.

MERS was first discovered in Saudi Arabia in 2012, and it presents severe respiratory symptoms such as high fever, cough and shortness of breath. In severe cases, it is accompanied by complications and even death. Although the clear source and route of infection have not been confirmed, it has been reported that infection is highly likely to be transmitted through contact with camels in the Middle East and transmission is possible through close person-to-person contact. The cases were mainly concentrated in the Middle East, but since May 2015, more than 100 cases have been reported across Korea.

As another human pathogen, human coronaviruses are known to cause the common cold. Among them, human coronavirus-OC43 is the most common human coronavirus and is known to cause upper respiratory tract infection. It seroconverts, and human coronavirus-OC43 was found to be the most common. Human coronavirus-OC43 infection has been shown to be involved in the development of severe respiratory diseases in children or the elderly, and has been confirmed to cause severe respiratory diseases in children (Pediatr. Infect. Dis. J., 32(4):325). -329).

As mentioned earlier, human coronavirus-OC43, MERS and SARS are all known variant coronaviruses belonging to the genus Beta coronavirus. Therefore, human coronavirus-OC43, MERS-coronavirus and SARS-coronavirus share similar characteristics in clinical symptoms, etiology and infection. In the phylogenetic tree, SARS-coronavirus belongs to strain B, which includes bat SARS-like coronaviruses and other bat-derived coronaviruses, and MERS-coronavirus belongs to strain C, which includes bat-derived coronaviruses. (Trends Microbiol 24:490-502). Furthermore, human coronavirus-OC43 belongs to lineage D formed by recombination between lineage B and lineage C.

Another human virus, SARS-CoV-2, is a new type of coronavirus (SARS-CoV-2) that first emerged in Wuhan, China in December 2019 and has spread throughout China and around the world. It is a respiratory infection caused by SARS-CoV-2 is transmitted when droplets (saliva) of an infected person penetrate the respiratory tract or the mucous membranes of the eyes, nose, and mouth. After an incubation period of about 2 to 14 days after infection, the main symptoms include fever (37.5 degrees), respiratory symptoms such as cough or respiratory difficulty, and pneumonia, but the frequency of asymptomatic infections is also high.

Since the antiviral agents developed to date show severe side effects, much attention is required in their application. These treatments are not effective and side effects are also present. Therefore, there is an increasing need for the development of excellent novel coronavirus agents with excellent infection suppression effect and low toxicity for preventing and treating coronavirus outbreaks.

In addition, in the case of a coronavirus composed of RNA genes, it is difficult to develop a therapeutic agent because antigens on the surface of the virus are constantly mutated, and it is difficult to predict which surface antigen will infect the human body.

Under this background, the present inventors have completed the present invention by confirming that as a result of continuing efforts to develop a novel coronavirus therapeutic agent, the Hyeoncho extract has an excellent effect of inhibiting the proliferation of coronavirus.

One object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of a coronavirus infection disease, comprising an extract of Hyeoncho as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for antiviral against coronavirus, comprising the extract of Hyeoncho as an active ingredient.

Another object of the present invention is to provide a preventive or therapeutic use of a coronavirus-infected disease, comprising a Hyeoncho extract as an active ingredient.

Another object of the present invention is to provide an antiviral use for coronavirus, comprising the extract of Hyeoncho as an active ingredient.

Another object of the present invention is to provide a method for preventing or treating a coronavirus-infected disease comprising administering the pharmaceutical composition to an individual.

Another object of the present invention is to provide a health functional food composition for the prevention or improvement of coronavirus-infected diseases comprising an extract of H. ginseng as an active ingredient.

Another object of the present invention is to provide an improved use of coronavirus-infected diseases comprising a Hyeoncho extract as an active ingredient.

Another object of the present invention is to provide a feed composition for the prevention or improvement of coronavirus infection disease, comprising the extract of Hyeoncho as an active ingredient.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be considered that the scope of the present invention is limited by the specific descriptions described below.

A first aspect of the present invention for achieving the above object provides a pharmaceutical composition for preventing or treating a coronavirus infection disease, comprising an extract of Hyeoncho as an active ingredient.

In addition, the second aspect of the present invention provides a pharmaceutical composition for antiviral against coronavirus, comprising the extract of Hyeoncho as an active ingredient.

In addition, a third aspect of the present invention provides a method for preventing or treating a coronavirus-infected disease comprising administering the pharmaceutical composition to an individual.

In addition, a fourth aspect of the present invention provides a preventive or therapeutic use of a coronavirus-infected disease, comprising an extract of Hyeoncho as an active ingredient.

In addition, a fifth aspect of the present invention provides an antiviral use for coronavirus, comprising a Hyeoncho extract as an active ingredient.

In addition, a sixth aspect of the present invention provides a health functional food composition for the prevention or improvement of a coronavirus infection disease comprising an extract of Hyeoncho as an active ingredient.

In addition, a seventh aspect of the present invention provides a feed composition for preventing or improving a coronavirus infection disease comprising a sagebrush extract as an active ingredient.

In addition, the eighth aspect of the present invention provides a use for improving coronavirus-infected diseases comprising an extract of Hyeoncho as an active ingredient.

Hereinafter, the present invention will be described in more detail.

As used herein, the term "hyeoncho" means that Geranium thunbergii Siebold et Zuccarini or other closely related plants (Geraniaceae) are collected before or when flowers bloom. Specifically, it is also called Nogwancho, and it is the above-ground part of the stem and the opposite leaves. Stems are thin and long, greenish-brown, with soft hairs on leaves and stems. The leaves are palm-shaped, divided into 3-5 branches, and are 2-4 cm long. It has a long petiole and is grayish-yellow-green to grayish-brown, and the lobe is long oval or obovate, with dull serrations. It has almost no odor and the taste is astringent.

As used herein, the term "extract" means a substance separated by a solvent from a liquid or solid mixture.

The Hyeoncho extract may be extracted with one or more solvents selected from water and alcohols having 1 to 6 carbon atoms. Specifically, the extraction solvent may include water, alcohol, benzene, ether, petroleum ether, ethyl acetate, or methanol.

As used herein, the term "coronavirus (Coronavirus)" refers to viruses belonging to the family Coronaviridae and is generally found in birds as well as various mammals including humans. It is one of the three major viruses that cause colds in humans. When viewed through an electron microscope, the surface of the virus particles protrude like a protrusion, and this shape is reminiscent of a crown or the corona of the sun. has been named Coronaviruses are diverse in their species, and depending on the nature and host of the virus, it means that it causes both respiratory and digestive infectious diseases. Specifically, recently, in humans, coronavirus is attracting attention as a causative agent that causes severe respiratory symptoms like SARS and MERS, which have been a worldwide problem.

The coronavirus may be SARS-CoV-2, but is not limited thereto.

As used herein, the term "SARS-CoV-2" is a respiratory infectious disease that first occurred in Wuhan, China in December 2019 and has spread throughout China and the world. Specifically, it was initially known only as a respiratory infectious disease of unknown cause, but on January 9, 2020, the World Health Organization (WHO) announced that the cause of the pneumonia was a new type of coronavirus (SARS-CoV-2, International Classification of Viruses). The pathogen was identified as the committee named it on February 11). It is a respiratory infection caused by a new type of coronavirus (SARS-CoV-2). is contagious when After an incubation period of about 2 to 14 days (estimated), the main symptoms are fever (37.5 degrees), respiratory symptoms such as cough or shortness of breath, and pneumonia, but the frequency of asymptomatic infections is also high.

As used herein, the term "coronavirus-infected disease" refers to symptoms or diseases caused by infection with a coronavirus. Specifically, body aches, sore throat, diarrhea, headache, conjunctivitis, loss of taste or smell, skin rash, discoloration of fingers or toes, difficulty breathing, chest pain, speech or movement disorders, cold, pneumonia, diabetes, cardiovascular disease, immune system damage, high blood pressure , renal failure, leukopenia, lymphopenia, nausea or vomiting, or respiratory disease.

As used herein, the term “prevention” refers to any act of inhibiting or delaying the onset of a virus by administering the composition according to the present invention.

As used herein, the term "improvement" refers to any action that at least reduces a parameter, for example, the severity of a symptom, associated with a condition to be treated by administration of a composition according to the present invention.

As used herein, the term "treatment" refers to any action in which the symptoms of a virus-infected individual are improved or beneficially changed by administration of the composition.

As used herein, the term "antiviral" refers to the action of inhibiting the proliferation of viruses. Specifically, the virus parasitizes in living cells of plants, animals, bacteria, and the like, and proliferates only in those cells. Depending on the type of nucleic acid they have, they are divided into DNA viruses and RNA viruses. Both are very small, less than a few hundred μm. Antivirus may be a prescription to prevent these viruses in advance, and may prevent the virus from reaching the onset even if it is infected.

Another embodiment of the present invention for achieving the above object provides a pharmaceutical composition for the prevention or treatment of coronavirus infection, or antiviral pharmaceutical composition comprising the extract of Hyeoncho as an active ingredient.

The composition of the present invention may further include a pharmaceutically acceptable salt.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent, wherein the carrier may include a non-naturally occurring carrier.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt in a form that can be used pharmaceutically among salts, which are substances in which a cation and an anion are combined by electrostatic attraction, and is usually a metal salt, an organic base and salts, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. For example, the metal salt may be an alkali metal salt (sodium salt, potassium salt, etc.), alkaline earth metal salt (calcium salt, magnesium salt, barium salt, etc.), an aluminum salt or the like; Salts with organic bases include triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N-dibenzylethylenediamine salts with, etc.; salts with inorganic acids may be salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like; Salts with organic acids may be salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like; Salts with basic amino acids may be salts with arginine, lysine, ornithine and the like; The salt with an acidic amino acid may be a salt with aspartic acid, glutamic acid, or the like.

Specifically, carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium Silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, polycaprolactone, polylactic acid Lactic Acid), poly-L-lactic acid, and mineral oil. The pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods. The form may include various amorphous carriers, microspheres, nanofibers, and the like. In the case of formulation, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract and its fractions, for example, starch, calcium carbonate, It may be prepared by mixing sucrose or lactose, gelatin, or the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid formulations for oral administration include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. The content of the Hyeoncho extract or a pharmaceutically acceptable salt thereof contained in the pharmaceutical composition of the present invention is not particularly limited.

Another embodiment of the present invention for achieving the above object provides a method for preventing or treating a coronavirus infection disease comprising administering the pharmaceutical composition to an individual.

At this time, the descriptions of "prevention", "antiviral", "coronavirus", "coronavirus infection disease", "prevention", "improvement" and "treatment" are the same as those described above.

As used herein, the term "individual" means all animals, such as rats, mice, livestock, etc., that have or can be caused by coronavirus infection. As a specific example, it may be a mammal.

As used herein, the term "administration" means introducing a predetermined substance into a subject by any suitable method, and the administration route of the pharmaceutical composition of the present invention is oral or parenteral through any general route as long as it can reach the target tissue. It can be administered orally. In addition, the pharmaceutical composition may be administered by any device capable of transporting the active agent to a target cell.

As used herein, the term “administration amount” may vary depending on the age, sex, and weight of an individual, and may be readily determined by those skilled in the art. Specifically, the pharmaceutical composition of the present invention may be administered orally or parenterally. In addition, the dosage of the pharmaceutical composition may be determined by a person skilled in the art according to the type of the composition as an active ingredient, along with several related factors such as the route of administration, the degree of disease, sex, weight, age, etc. The scope is not limited.

As used herein, the term "administration method" refers to various methods such as oral, intravenous, subcutaneous, intradermal, intranasal, intraperitoneal, intramuscular, and transdermal.

Another embodiment of the present invention for achieving the above object provides a health functional food composition for the prevention or improvement of coronavirus infection disease, or antiviral, comprising the extract of sagebrush as an active ingredient.

The composition of the present invention may further include a food pharmaceutically acceptable salt.

Another embodiment of the present invention for achieving the above object is that the extract according to the present invention exhibits an excellent prevention or treatment effect of a coronavirus infection disease, and thus a food composition for the purpose of preventing or treating a coronavirus infection disease, antiviral It may be included in, and since the food composition can be consumed on a daily basis, a high effect can be expected for the prevention or treatment of antiviral, coronavirus-infected diseases.

At this time, the descriptions of "prevention", "antiviral", "coronavirus", "coronavirus infection disease", "prevention", "improvement" and "treatment" are the same as those described above.

As used herein, the term “health functional food” refers to food manufactured and processed using raw materials or ingredients useful for the human body in accordance with Act No. 6727 of the Health Functional Food Act, and 'functionality' refers to the structure of the human body. And it means to obtain a useful effect for health use, such as regulating nutrients for function or physiological action. On the other hand, health food means food that has an active health maintenance or promotion effect compared to general food, and health supplement means food for the purpose of health supplementation. In some cases, the terms health functional food, health food and health supplement food can be mixed.

The extract of the present invention or a pharmaceutically acceptable salt thereof may be added as it is or may be used with other foods or food ingredients, and may be appropriately used according to a conventional method.

The food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art. Specifically, the food composition may further include a physiologically acceptable carrier, the type of carrier is not particularly limited and any carrier commonly used in the art may be used. In addition, the food composition contains food additives such as preservatives, disinfectants, antioxidants, colorants, coloring agents, bleaching agents, seasonings, sweeteners, flavoring agents, expanding agents, strengthening agents, emulsifying agents, thickeners, filming agents, gum base agents, foam inhibitors, solvents, and improving agents. may include The additive may be selected according to the type of food and used in an appropriate amount.

In addition, the formulation of the food may be prepared without limitation as long as it is a formulation recognized as a food. The food composition of the present invention can be prepared in various types of dosage forms, and unlike general drugs, it has the advantage of not having side effects that may occur during long-term administration of drugs by using food as a raw material, and is excellent in portability. Food can be ingested as a supplement to enhance the effectiveness of antiviral or coronavirus-infected disease prevention or treatment.

Hyeoncho extract of the present invention or a pharmaceutically acceptable salt thereof may be included in various weight % in the food composition if it can exhibit antiviral and coronavirus-infected disease prevention or treatment effects. Specifically, it may be included in an amount of 0.00001 to 100% by weight or 0.01 to 80% by weight relative to the total weight of the food composition, but is not limited thereto. In the case of long-term ingestion for health and hygiene purposes, it may contain an amount below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

Another embodiment of the present invention for achieving the above object provides a feed composition for the prevention or treatment of antiviral, or coronavirus-infected disease comprising the extract of Hyeoncho as an active ingredient.

The composition of the present invention may further include a food pharmaceutically acceptable salt.

At this time, the descriptions of "prevention", "antiviral", "coronavirus", "coronavirus infection disease", "prevention", "improvement" and "treatment" are the same as those described above.

Since the Hyeoncho extract according to the present invention exhibits excellent antiviral, preventive and therapeutic effects of coronavirus infection diseases, it may be included in a feed composition for the purpose of preventing and treating antiviral and coronavirus infection diseases, the feed composition comprising Since it is possible for animals to consume it on a daily basis, a high effect can be expected for the prevention or treatment of coronavirus-infected diseases and antiviral.

As used herein, the term "feed" means any natural or artificial diet, meal, etc., or a component of said meal, intended for or suitable for being eaten, consumed, and digested by an animal. The type of feed is not particularly limited, and feed commonly used in the art may be used. Non-limiting examples of the feed include plant feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, gourds or grain by-products; and animal feeds such as proteins, inorganic materials, oils and fats, minerals, oils and fats, single cell proteins, zooplankton or food. These may be used alone or in mixture of two or more.

As shown in FIG. 1 , the Hyeoncho extract of the present invention inhibits the binding between the spike protein of SARS-CoV-2 to the human ACE2 protein receptor, and thus can suppress the infection of the coronavirus.

As used herein, the term "Angiotensin-converting enzyme 2 (ACE2) protein" is one of metallo-carboxypeptidase, a type 1 transmembrane protein homologous to angiotensin converting enzyme, and is a eukaryotic It can be found in living organisms and bacteria.

As used herein, the term "spike protein" refers to a dendritic structure arranged on the surface of the outer skin. Specifically, although it is difficult to morphologically distinguish between the two on the surface of the virus particle, if each spike is isolated and purified, the morphological difference can be recognized, and the spike plays an essential role in adsorption and invasion of the enveloped virus into the host cell. .

Coronavirus is an important weapon in which the spike protein that protrudes like a crown on the surface of the virus invades humans. Angiotensin-converting enzyme 2 in the body is a receptor protein that binds to the spike protein of coronavirus to invade the body and cause infection. Angiotensin-converting enzyme 2, a protein in the body, has a function of preventing lung damage. When the coronavirus spike protein binds to the receptor of ACE2 and the virus penetrates the body, severe acute respiratory syndrome may occur.

In a specific example, it was confirmed that the Hyeoncho extract of the present invention effectively inhibits the binding between ACE2 and the coronavirus spike protein.

Therefore, through the specific example above, it was confirmed that the Hyeoncho extract of the present invention has an effect of inhibiting the binding between the ACE2 protein and the spike protein of the coronavirus because the ACE2 protein is the passage through which the coronavirus penetrates, which furthermore is a coronavirus infection disease. It suggests that it can be used for the prevention or treatment of, or antiviral use.

Since the extract of the present invention exhibits an effect of inhibiting infection of coronaviruses such as SARS-CoV-2 (SARS-CoV-2), it can be used to develop antiviral agents for preventing or treating the viral diseases.

1 is a schematic diagram showing the binding relationship between the spike protein of SARS coronavirus-2 (SARS-CoV-2) and human ACE2 protein.

2 is a graph showing the inhibition rate of binding between the spike protein of SARS-CoV-2 (SARS-CoV-2) and the human ACE2 protein according to the concentration of the Hyeoncho extract of the present invention.

3 is a photograph showing the inhibition of cell infection of the Hyeoncho extract of the present invention.

4 is a graph showing the infection inhibition rate and cell viability for SARS-CoV-2 (SARS-CoV-2) in Hyeoncho Chuchummul of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These Examples are for explaining the present invention in more detail, and the scope of the present invention is not limited by these Examples.

Preparation Example 1: Method for preparing an extract of Hyeoncho

After drying 10 g of Huncho (Geranium thunbergii Siebold et Zuccarini), 100 ml of 30-70% (v/v) ethanol was added as a solvent, extracted under reflux three times for 2 hours, and concentrated under reduced pressure to prepare a Hyeoncho extract.

Example 1: Confirmation of inhibition of binding to the spike protein of SARS-CoV-2 (SARS-CoV-2) of the extract of Hyeoncho

In order to confirm the SARS-CoV-2 infection inhibitory ability of the extract of Hyeoncho, the binding inhibition rate between the spike protein of ACE2 and SARS-CoV-2 (SARS-CoV-2) according to the concentration was confirmed.

Specifically, in order to confirm the inhibition of binding between human ACE2 protein and the spike protein of SARS-CoV-2, Hyeoncho extract by concentration (1.56, 3.12, 6.25, 12.5, 25, 50, 100 μg) /ml) was added to confirm the binding inhibition rate. In this case, anti-spike proteins (6.25, 12.5, 25, 50, 100 ng) were used as a positive control.

As shown in Figure 2, the Hyeoncho extract of the present invention inhibited the binding between the spike protein of human ACE2 protein and SARS-CoV-2 (SARS-CoV-2) even in the smallest amount of 1.56 μg / ml, positive It was confirmed that the binding inhibition rate of the Hyeoncho extract compared to the control was excellent, and the binding was inhibited in a concentration-dependent manner.

Therefore, it was confirmed that the Hyeoncho extract of the present invention has antiviral activity against SARS-CoV-2 (SARS-CoV-2).

Example 2: Inhibition of cell infection of Hyeoncho extract

In a 24-well TC plate, hACE2/TMPRSS2 overexpressing HEK293 cells (1Х105 cells/well) were inoculated (cell seeding). After 18-20 hours, SARS-CoV-2 spike-like lentivirus (SARS-CoV-2 Spike) expressing GFP-expressing SARS-CoV-2 spike protein antibody (1 μg/ml) or SARS-CoV-2 spike protein antibody (SARS-CoV-2 Spike) -Pseudotyped Lentivirus) (MOI: 1) at 37 °C for one hour, and then inoculated into hACE2/TMPRSS2-overexpressing HEK293 cells. After 2 hours of inoculation with SARS-CoV-2 GFP virus with Hyeoncho extract, the inoculum was removed from the cells. After washing 3 times with PBS, it was replaced with DMEM containing 10% FBS. A negative control was treated only with DMEM supplemented with 10% FBS. As a positive control, SARS-CoV-2 spike antibody was used. After 72 hours, the degree of virus infection was checked using a fluorescence microscope to confirm the inhibition of virus infection.

As shown in FIG. 3 , the virus infection tested by immunofluorescence by SARS-CoV-2 infection in cells was detected in the cytoplasm of the cells, but the Hyeoncho extract of the present invention did not detect SARS-CoV-2 infection in the cells. , it was confirmed that it exhibits an antiviral effect.

Example 3: Confirmation of infection inhibition of SARS-CoV-2 (SARS-CoV-2) of Hyeoncho extract

Cell-based in vitro antiviral efficacy was evaluated in order to confirm that the extract of sagebrush actually inhibits the infection of SARS-CoV-2 (Wuhan coronavirus-19, provided by the Korea Centers for Disease Control and Prevention). The antiviral efficacy of the compound was determined by a dose response curve (DRC) experiment in a cell infection model of SARS-CoV-2.

1.2 Х 104 Vero cells (ATCC-CCL81) per well were inoculated into a 384-tissue culture plate, and 24 hours later, a sample obtained by serially diluting the Hyeoncho extract twice was treated for about 1 hour. Then, SARS-CoV-2 (0.0125 MOI) was infected and cultured for 24 hours. Thereafter, cells were fixed with 4% paraformaldehyde (PFA), permeabilized, and treated with anti-SARS-CoV-2 nucleocapsid (N) primary antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody, Hoechst 33342. was dyed. Fluorescence images of infected cells were acquired using Operetta (Perkin Elmer) and analyzed using Columbus software. The total number of cells per well was calculated as the number of nuclei stained with Hoechst, and the number of infected cells was calculated as the number of cells expressing the viral N protein. The infectivity (infection ratio) was calculated as the number of cells expressing the N protein/total number of cells. The infectivity per well was normalized to the average infectivity of wells containing uninfected cells (mock) and the average infectivity of wells containing infected cells treated with 0.5% DMSO (v/v) in the same plate. The cell viability of the compound was expressed as 'cell viability' on the graph by normalizing the number of cells in each well to the average number of wells in the mock group (Fig. 4).

As shown in FIG. 4 , it was confirmed that the excellent inhibitory ability of SARS coronavirus-2 (SARS-CoV-2) against Vero cells of the Hyeoncho-derived compound was confirmed, and the cell viability was also maintained high.

Summarizing Examples 1 to 3 of the present invention, the Hyeoncho extract of the present invention inhibits the binding between human ACE2 protein and the spike protein of SARS-CoV-2, SARS-CoV-2 It can be confirmed that there is an antiviral effect on 2 (SARS-CoV-2). Furthermore, by experimentally verifying the significant SARS-CoV-2 infection inhibitory effect and high cell viability of the compound derived from Hyeoncho, it was suggested that the composition of the present invention can be used as a useful therapeutic agent for the above viral diseases.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the following claims and their equivalents.

Claims (6)

1. A pharmaceutical composition for the prevention or treatment of coronavirus-infected diseases, comprising the extract of Hyeoncho as an active ingredient.
2. According to claim 1,

   The coronavirus is SARS-CoV-2 (SARS-CoV-2), the pharmaceutical composition.
3. A pharmaceutical composition for antiviral against coronavirus, comprising the extract of Hyeoncho as an active ingredient.
4. A method of preventing or treating a coronavirus-infected disease comprising administering the pharmaceutical composition of claim 3 to an individual.
5. A health functional food composition for the prevention or improvement of coronavirus-infected diseases, comprising an extract of sagebrush as an active ingredient.
6. A feed composition for preventing or improving coronavirus-infected diseases, comprising an extract of Hyeoncho as an active ingredient.

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