CN116963754A - A composition for preventing or treating coronavirus infection comprising an extract of Hovenia truncatula - Google Patents

Abstract

The present application relates to an antiviral use against coronavirus comprising hovenia dulcis thunb extract or an isolate thereof, and more particularly, to an antiviral pharmaceutical composition against coronavirus comprising hovenia dulcis thunb extract or an isolate thereof, a pharmaceutical composition for preventing or treating infectious diseases of coronavirus, and a method for preventing or treating infectious diseases of coronavirus using the same, a health functional food composition for preventing or improving infectious diseases of coronavirus comprising hovenia dulcis thunb extract or an isolate thereof, and a feed composition for preventing or improving infectious diseases of coronavirus comprising hovenia dulcis thunb extract or an isolate thereof. Since the hovenia dulcis thunb extract of the present application exhibits an effect of inhibiting infection of coronaviruses such as MERS-coronavirus, SARS-coronavirus and SARS-coronavirus-2, it is useful for developing antiviral agents for preventing or treating the above-mentioned viral diseases.

Description

A composition containing an extract of Hovenia truncatula for preventing or treating coronavirus infection compound

Technical field

The present invention relates to an antiviral use against coronavirus comprising an extract of Hovenia truncatula or its isolate.

Background technique

After the coronavirus was first discovered in chickens in 1937, it subsequently passed through animals such as dogs, pigs, and birds, and was also discovered in humans in 1965. During a total solar eclipse, when the sun's light ball is covered by the moon, a white glow appears around it, which is similar to the coronavirus, hence the name.

Coronaviruses are known to mainly cause pneumonia and enteritis in humans and animals, and occasionally cause neurological infections and hepatitis. Coronaviruses belong to the Coronaviridae family, have a spherical outer membrane, and are positive senseRNA viruses with a size of approximately 100-120 nm (Master, 2006). There are five structural proteins of coronavirus: the outermost spike protein (S), hemagglutinin-esterase (HE) protein, transmembrane (M) protein, small membrane (E) protein and nucleocapsid (N) Protein (Lai and Homes, 2001. Fields Virology). Among them, the spike protein acts as a ligand that binds to cell receptors and is a protein that induces the fusion of host cells and viruses. It is considered to be the most mutated protein.

So far, coronaviruses are considered pathogens that rarely infect humans and mainly infect animals such as dogs, pigs, and cattle. Even if it infects humans, it is just one of many viruses that cause respiratory symptoms, cause the common cold, or cause intestinal illnesses such as diarrhea, which are less dangerous to children. However, people are gradually paying attention as the pathogen of Middle East Respiratory Syndrome (MERS), which has caused hundreds of deaths and thousands of patients around the world, is a new (mutated) coronavirus.

COVID-19 is a disease causing a progressive pandemic and is an infectious disease caused by SARS-coronavirus-2. As of May 7, 2020, 187 countries have reported more than 3.75 million cases, 263,000 deaths, and 1.24 million recoveries. Common symptoms are fever, cough, fatigue, shortness of breath, and loss of smell and taste. In most cases, symptoms are mild, but some develop into viral pneumonia, multi-organ failure, and cytokine storm. The time from symptom onset to onset is generally about 5 days, but can range from 2 to 14 days. The virus spreads mainly from person to person through close contact and sometimes through droplets produced when coughing, sneezing and talking. It is most contagious within the first 3 days after symptoms appear and can be spread before symptoms appear or even at the end of the illness. The standard way to diagnose it is real-time reverse transcription polymerase chain reaction (rRT-PCR) using a nasopharyngeal swab.

MERS was first discovered in Saudi Arabia in 2012. It presents with severe respiratory symptoms such as high fever, cough, and difficulty breathing. It is a disease that is accompanied by complications and may lead to death in severe cases. Although the exact source and route of infection have not yet been identified, it is reported that infection is very likely through contact with camels in the Middle East and may be spread through close contact between people. Patients are mainly distributed in the Middle East, but since May 2015, more than 100 infected people have appeared across South Korea.

As mentioned earlier, both MERS and SARS are known to be caused by mutant coronaviruses (MERS-coronavirus, SARS-coronavirus, and SARS-coronavirus-2) belonging to the genus Betacoronavirus. Therefore, MERS-coronavirus, SARS-coronavirus, and SARS-coronavirus-2 have similar characteristics in terms of clinical symptoms, pathogenesis, and infection. In the phyloge netictree, SARS-coronavirus and SARS-coronavirus-2 belong to the B lineage including bat SARS-like coronaviruses and other bat-derived coronaviruses, while MERS-coronavirus belongs to the B lineage including bat-derived coronaviruses. Lineage C of the virus (Trends Microbiol 24:490-502).

MERS-coronavirus, SARS-coronavirus and SARS-coronavirus-2 are all thought to originate from bats, which are the main carriers of various coronaviruses (Antiviral Res 101:45-56), and their exact infection routes have not yet been fully elucidated. On the other hand, cross-species transmission of the virus to palm civets (palm ci vet) and dromedary camels (dromedary camel) increases the possibility of zoonotic infections (Nat Rev v Microbiol 14:523-534), and nosocomial infections are considered It is the main cause of human-to-human transmission of MERS coronavirus and SARS-coronavirus infections (BMC Med13:1-12).

On the other hand, although several drugs are currently being developed to treat SARS-coronavirus-2, they are mainly based on chemical compounds or existing drugs, and natural drugs have not yet been developed.

In particular, since the antiviral drugs developed to date have serious side effects, great caution is required when applying them. In fact, this treatment was ineffective and had side effects. Therefore, there is an increasing need to develop novel coronavirus drugs with excellent infection inhibition effects and low toxicity to prevent and treat coronaviruses.

Against this background, the inventors have made unremitting efforts in the research and development of new coronavirus therapeutic drugs and discovered that the Hovenia dulcis extract with guaranteed stability can effectively combat SARS-coronavirus-2, SARS-coronavirus and MERS-coronavirus. of proliferation. It was confirmed that it has an excellent inhibitory effect, and the present invention was completed.

Contents of the invention

The inventors identified for the first time the antiviral effect of Hovenia vesifolia extract or its isolate against coronavirus, and developed an antiviral pharmaceutical composition for coronavirus containing the Hovenia vesifolia extract or its isolate. Pharmaceutical compositions for preventing or treating coronavirus infectious diseases, methods for preventing or treating coronavirus infectious diseases using the above-mentioned pharmaceutical compositions, and methods for preventing or ameliorating coronavirus infectious diseases containing Hovenia mandarin extracts or isolates thereof The present application is completed with a health functional food composition and a feed composition containing Hovenia dulcis extract or its isolate for preventing or improving coronavirus infectious diseases.

One object of the present invention is to provide an antiviral pharmaceutical composition against coronavirus containing an extract of Hovenia truncatula or its isolate.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating coronavirus infectious diseases containing an extract of Hovenia truncatula or its isolate.

Another object of the present invention is to provide a method for preventing or treating coronavirus infectious diseases using the above pharmaceutical composition.

Another object of the present invention is to provide a health functional food composition for preventing or ameliorating coronavirus infectious diseases containing an extract of Hovenia truncatula or its isolate.

Another object of the present invention is to provide a feed composition for preventing or improving coronavirus infectious diseases containing an extract of Hovenia truncatula or its isolate.

Another object of the present invention is to provide the use of a composition against coronavirus, which is used to manufacture an antiviral pharmaceutical composition, including an extract of Hovenia truncatula or an isolate thereof.

Another object of the present invention is to provide the use of a composition for manufacturing a pharmaceutical composition for preventing or treating coronavirus infectious diseases, including an extract of Hovenia truncatula or its isolate.

Another object of the present invention is to provide the use of a composition for manufacturing a health functional food composition for preventing or improving coronavirus infectious diseases, including an extract of Hovenia truncatula or its isolate.

Another object of the present invention is to provide the use of a composition for manufacturing a feed composition for preventing or improving coronavirus infectious diseases, including an extract of Hovenia truncatula or its isolate.

Effect of the invention

Since the Hovenia truncatula extract of the present invention exhibits the effect of inhibiting coronavirus infections such as SARS-coronavirus-2, SARS-coronavirus and MERS-coronavirus, it can be used to develop antimicrobials for preventing or treating the above viral diseases. Viral agent.

Description of the drawings

Figure 1 is a graph showing the SARS-coronavirus-2 inhibitory efficacy of the concentration of the Hovenia dulcis extract according to the present invention and the control group remdesivir.

best practice

In the following, the invention will be described in more detail.

In one aspect, each description and embodiment disclosed in this application may also apply to each different description and embodiment. That is, all combinations of the elements disclosed in this application belong to the scope of the present invention. Furthermore, the scope of the present invention is not limited by the following detailed description.

Furthermore, those of ordinary skill in the art can identify or confirm, using no more than routine experimentation, most equivalents to the specific forms of the invention described herein. Furthermore, such equivalents are intended to be encompassed by the present invention.

In order to achieve the above object, one embodiment of the present invention provides an antiviral pharmaceutical composition for coronavirus containing an extract of Hovenia truncatula or its isolate.

In order to achieve the above object, another embodiment of the present invention provides a pharmaceutical composition for preventing or treating coronavirus infectious diseases that contains an extract of Hovenia truncatula or its isolate as an active ingredient.

Specifically, since the pharmaceutical composition of the present invention exhibits the effect of inhibiting coronavirus, the present invention can be effectively used to prevent or treat infectious diseases caused by coronavirus.

The term "Hove Citrus" in the present invention refers to the fruits or seeds of Hovenia trifoliata with fruit stalks in the family Rhamnaceae. Because of its sweet taste, it is called wood honey, which means honey growing on trees. In China, it is also called Xuanpu pear, which means the pear tree growing in the fairy garden. Because it is as white and hard as stone, it is also called white stone wood.

It is used for fever, thirst, burping, vomiting, etc. caused by fever, and is known to be a diuretic, treat liver damage caused by alcoholism, promote blood circulation, and relax muscles. In addition, Korean Patent No. 0588759 discloses a food composition containing Hovenia truncatula for improving cognition and a health functional food using the composition, and Korean Patent No. 1314749 discloses a food composition containing Hovenia truncatula tree and Korean registered patent No. 1306878 discloses a method for producing a composition for tea bags of Hovenia vesifolia, a composition for preventing and treating bone diseases containing a Hovenia vesifolia extract as an active ingredient. However, so far, there are no reports on the antiviral activity of using Hovenia dulcis extract against coronavirus.

As mentioned above, the term "extract" in the present invention has the meaning commonly used in the art as a crude extract, but in a broad sense also includes isolates obtained by further separation of the crude extract. That is, the Hovenia dulcis extract of the present invention includes not only the extract obtained using the above-mentioned extract solvent but also the isolate obtained by additionally applying a purification process thereto. For example, separations obtained by passing the extract through an ultrafiltration membrane with a certain molecular weight cutoff, through various chromatographic separations (designed to separate based on size, charge, hydrophobicity, or affinity), separations obtained by the above extract solvents Separation by various additional purification methods, such as by secondary or tertiary additional extractions, is also included in the extracts of the present invention.

In the Hovenia vesifolia extract of the present invention, the method of extracting Hovenia vesifolia is not particularly limited and can be extracted according to conventional methods in this field. Non-limiting examples of the above extraction methods include: hot water extraction method, ultrasonic extraction method, filtration method, reflux extraction method, etc. These methods can be performed alone or in combination of two or more methods.

In the present invention, the type of solvent used for the extract is not particularly limited, and any solvent known in the art can be used. Non-limiting examples of extract solvents include water, alcohol or mixed solvents thereof, etc., which can be used alone or in combination of one or more. Specifically, water, methanol, ethanol or butanol can be used. When alcohol is used as a solvent, alcohols having 1 to 4 carbon atoms can be specifically used.

For example, Hovenia dulcis extract can be extracted by a hot water extraction method including the following steps: Step 1) Add 5-30 times of distilled water based on the weight of Hovenia vesifolia; Step 2) At a temperature of 100-130°C Add the hot water extract for 2 to 5 hours; step 3) filter the hot water extract; but it is not limited to this.

The term "coronavirus infectious disease" in the present invention refers to a disease caused by coronavirus invading the body of a host organism and infecting it. Specifically, the above-mentioned coronavirus can be selected from the group consisting of SA RS-coronavirus-2 (coronavirus- 19), more than one of the group consisting of SARS-coronavirus and MERS-coronavirus, more specifically, it can be SARS-coronavirus-2.

The term "SARS-coronavirus-2" as used herein is a strain of the virus that causes the respiratory disease coronavirus disease 2019 (COVID-19), as specified by the National Health Service (Korea) It is known to the National Institutes of Health (NIH) as the successor of SARS-coronavirus. SARS-coronavirus-2 is a benign single-stranded RNA virus (positive-sense single-stranded RNA virus). It is contagious in humans, and the World Health Organization (World Health Organization; WHO) has declared the ongoing COVID-19 pandemic a Public Health Emergency of International Concern (PHEIC) ).

SARS-CoV-2 is taxonomically considered a strain of SARS-CoV, has zoonotic origins, and has close genetic similarity to bat coronaviruses. The above-mentioned SARS-coronavirus-2 is mainly spread through close contact between people and/or through droplets produced when coughing or sneezing, and mainly interacts with the receptor angiotensin converting enzyme 2 (ACE2) ) combines into human cells.

The term "SARS-coronavirus (SARS-coronavirus)" of the present invention is an ssRNA virus belonging to the genus βcoronavirus with an envelope. Its entire genome consists of 29,727 nucleotides, making it the largest genome among RNA viruses known to date. The genome of SARS-CoV is known to have 11 open reading frames (ORFs) encoding 23 proteins. It was found that the main structural proteins of SARS-CoV are nucleocapsid (N), spike (S), membrane (Membrane, M) and small envelope (E) proteins.

After sequencing these proteins, it was found that compared with other coronaviruses, the sequence homology of SARS-coronavirus is very low, about 40% to 50%. According to the phylogenetic classification of coronaviruses, it was found that SARS coronavirus has a higher affinity with group II among groups I, II, and III.

The term "MERS-coronavirus" of the present invention is a newly discovered ssRNA virus belonging to the genus Betacoronavirus on September 24, 2012. It has a 5'-replication enzyme-structural protein (spikeenvelope-membrane-nucleocapsid). )-poly(A)-3′[5′-ORF1a/b-S-E-M-N-poly(A)] structure. The MERS-coronavirus genome is phylogenetically divided into clade A and clade B. The early MERS cases were clade A (EMC/2012 and Jordan-N3/2012), and the newly reported cases are of genetically different clade B ( Emerging Infectious Diseases, Vol. 20, No. 6, June 2014).

In the present invention, a pharmaceutical composition for preventing or treating coronavirus infectious diseases containing the Hovenia truncatula extract or its isolate is characterized by inhibiting coronavirus infectious diseases, such as SARS-coronavirus-2, One or more viruses selected from the group consisting of SARS-coronavirus and MERS-coronavirus. Specifically, in one embodiment of the present invention, it was confirmed that the above-mentioned Hovenia truncatula extract or its isolate has the ability to inhibit SARS-coronavirus-2 infection, and was compared with the positive control group Remdesivir (figure 1).

The term "infectious disease" in the present invention refers to diseases caused by infection.

The above term "infection" refers to the state in which pathogenic microorganisms invade the host organism and grow and reproduce.

The term "prevention" of the present invention refers to all actions to inhibit or delay the onset of infectious diseases caused by coronavirus by administering the pharmaceutical composition according to the present invention.

The term "treatment" of the present invention refers to all actions that improve or beneficially change the symptoms of individuals with suspected or known coronavirus infectious diseases through the administration of pharmaceutical compositions.

The pharmaceutical composition of the present invention may also include pharmaceutically acceptable carriers, excipients or diluents, and the above-mentioned carriers may include non-naturally occurring carriers.

More specifically, as carriers, excipients and diluents that can be included in the above-mentioned pharmaceutical compositions, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, Maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate Ester, talc, magnesium stearate, polycaprolactone (polycaprolactone), polylactic acid (Poly Lactic Acid), poly-L-lactic acid (poly-L-lactic acid), mineral oil.

The above-mentioned pharmaceutical compositions can be prepared according to conventional methods into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and external dosage forms, suppositories, and sterile injections. For use, the form of carriers can include: various irregular carriers, microspheres, nanofibers, etc.

When formulating, commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants and other diluents or excipients can be used for preparation.

Solid preparations for oral administration include: tablets, pills, powders, granules, capsules, etc. This solid preparation can be prepared by mixing the above extract and its isolate with at least one excipient such as starch, calcium carbonate It is prepared by mixing calcium carbonate, sucrose or lactose, gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium styrenate and talc can also be used.

Liquid preparations for oral administration include: suspensions, oral solutions, emulsions, syrups, etc. In addition to common simple diluents such as water and liquid paraffin, they can also include wetting agents, sweeteners, aromatics, Preservatives and other excipients.

Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, adjuvants, and the like. Examples of non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

There is no particular restriction on the content of the Hovenia vesifolia extract in the pharmaceutical composition of the present invention, and a pharmaceutically effective amount of the Hovenia vesifolia extract may be included.

The term "pharmaceutically effective dose" of the present invention refers to an amount sufficient to treat a disease with an effective dose/risk ratio suitable for medical treatment, and is usually an amount of 0.001 to 1000 mg/kg administered once to several times a day, An amount of 0.05 to 200 mg/kg is preferred, and an amount of 0.1 to 100 mg/kg is more preferred. However, for the purposes of this invention, the effective amount for a particular treatment in a particular patient will preferably depend on the type and degree of response to be achieved, the particular composition including whether other agents are used as appropriate, the age, weight, general health of the patient. Condition, gender and diet, time of administration, route of administration and secretion rate of the composition, duration of treatment, various factors including drugs used with or concurrently with a particular composition, and similar factors well known in the medical field, may vary in the method of application.

The pharmaceutical composition of the present invention can be used as a single therapeutic agent or in combination with other therapeutic agents, and can also be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered single or multiple times. Taking into account all the above factors, it is important to administer an amount that can obtain the maximum effect in the minimum amount without causing side effects, and can be easily determined by a person skilled in the art.

The term "administration" of the present invention refers to introducing the pharmaceutical composition of the present invention into a patient by any appropriate method, and the administration route of the composition of the present invention can be through various oral or non-oral routes, as long as the target can be reached. Just organize.

The administration method of the pharmaceutical composition according to the present invention is not particularly limited and can be carried out according to methods commonly used in the art. As non-limiting examples of administration methods, the composition may be administered by oral administration or parenteral administration. The pharmaceutical composition according to the present invention can be prepared into various dosage forms according to the target administration mode.

The dosage of the pharmaceutical composition of the present invention can be, for example, 1 to 20 mg/kg per day, more preferably 1 to 10 mg/kg per day, to mammals including humans. . The administration frequency of the composition of the present invention is not particularly limited. It may be administered once a day or several times in divided doses.

In order to achieve the above object, another embodiment of the present invention provides a method for preventing or treating coronavirus infectious diseases, including administering a pharmaceutical composition to people other than those who have symptoms of or are at risk of coronavirus infectious diseases. Individual steps.

At this time, the descriptions of “pharmaceutical composition”, “coronavirus”, “infection”, “prevention” and “treatment” are as described above.

The term "individual" in the present invention may refer to all animals including humans who have symptoms of coronavirus infection or are at risk of developing the disease. The animal can be not only humans, but also cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, cats and other mammals that need treatment due to similar symptoms, but are not limited thereto.

Specifically, the above-mentioned prevention or treatment method of the present invention may include the step of administering a pharmaceutically effective amount of the composition to individuals who have symptoms of coronavirus infection or are at risk of developing the disease.

The term "administration" of the present invention refers to introducing the pharmaceutical composition of the present invention into an individual with symptoms of coronavirus infection or at risk of developing the disease by any appropriate method, and the route of administration of the composition of the present invention can be oral or parenteral. Various routes of oral administration are sufficient as long as it can reach the target tissue.

In order to achieve the above object, another embodiment of the present invention provides a health functional food composition for preventing or improving coronavirus infectious diseases containing an extract of Hovenia truncatula or its isolate.

At this time, the descriptions of “Hovenia dulcis extract”, “its isolate”, “coronavirus”, “infection” and “prevention” are as described above.

Since the Hovenia dulcis extract according to the present invention exhibits excellent inhibitory effects on coronavirus, it can be included in a food composition to prevent or improve infectious diseases caused by coronavirus, since the above food composition can be consumed daily. , so it can be expected to have a high effect in preventing or improving coronavirus infection.

The term "improvement" according to the invention refers to all actions that reduce at least the degree of parameters, for example symptoms, related to the condition treated by the administration of the pharmaceutical composition according to the invention.

The term "health functional food" in the present invention refers to food that is manufactured and processed using raw materials or ingredients that are beneficial to the human body in accordance with the Health Functional Food Act No. 6727. "Function" refers to the effect on the structure and function of the human body such as nutritional regulation or Physiological effects and other beneficial health effects. On the other hand, health food refers to food that has a positive effect in maintaining or promoting health compared with general food. Health supplement food refers to food with the purpose of supporting health. Depending on the situation, health functional food, health food, health supplement food The terms can be used interchangeably.

The Hovenia dulcis extract of the present invention can be added directly or used together with other foods or food ingredients, and can be used appropriately according to conventional methods.

The food of the present invention can be prepared by methods commonly used in the art, and can be prepared by adding raw materials and ingredients commonly used in the art during the above preparation process. Specifically, the above-mentioned food composition may also 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 above food composition may include preservatives, bactericides, antioxidants, colorants, coloring agents, bleaching agents, flavoring agents, sweeteners, spices, swelling agents, enhancers, emulsifiers, thickeners, coatings agents, gum bases, foam inhibitors, solvents, improvers and other food additives. The above additives can be used in appropriate amounts according to the type of food.

In addition, the dosage form of the above-mentioned food can be produced without restriction as long as it is a dosage form considered to be a food. The food composition of the present invention can be made into various dosage forms. Unlike general medicines, since it uses food as a raw material, it has the advantage of not having side effects that may occur when taking medicines for a long time, and it has excellent portability. Therefore, the food composition of the present invention Supplement intake that can be used to enhance the effect of preventing or ameliorating infectious diseases caused by coronavirus.

If the Hovenia dulcis extract of the present invention can show the effect of preventing or ameliorating infectious diseases caused by coronavirus, it can be included in the food composition in various weight %. Specifically, it may include 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, a content lower than the above range may be contained, and since there is no problem in terms of safety, an amount of the active ingredient above the above range may be used.

In order to achieve the above object, another embodiment of the present invention provides a feed composition for preventing or ameliorating coronavirus infectious diseases that contains a Hovenia truncatula extract or an isolate thereof as an active ingredient.

At this time, the descriptions of “Hovenia dulcis extract”, “isolate thereof”, “coronavirus”, “infectious disease”, “prevention” and “improvement” are as described above.

Since the Hovenia dulcis extract according to the present invention exhibits an excellent inhibitory effect on coronavirus, it is included in a feed composition for the purpose of preventing or improving coronavirus infectious diseases, and animals can daily ingest the above-mentioned feed composition, so it can It is expected to be highly effective in preventing or ameliorating infectious diseases caused by coronavirus.

The term "feed" as used herein means any natural or artificial prescribed meal, meal, etc. or ingredients thereof, intended for consumption, ingestion and digestion by animals, or suitable for that purpose.

The types of the above-mentioned feeds are not particularly limited, and feeds commonly used in this field can be used. Non-limiting examples of the above-mentioned feeds include: plant-based feeds such as cereals, dried fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils, starches, meal or cereal by-products; such as protein, inorganic Physical, organic, mineral, organic, single-cell protein, animal float or, physical and other animal feed. These can be used individually or in mixture of 2 or more types.

Another object of the present invention is to provide the use of a composition containing an extract of Hovenia truncatula or its isolate in the preparation of an anti-coronavirus pharmaceutical composition.

At this time, the descriptions of “Hovenia dulcis extract”, “its isolate” and “coronavirus” are as described above.

Another object of the present invention is to provide the use of a composition containing Hovenia truncatula extract or its isolate in the preparation of pharmaceutical compositions for preventing or treating coronavirus infectious diseases.

At this time, the descriptions of "Philipium foliage extract", "its isolate", "coronavirus", "infectious disease", "prevention" and "treatment" are as described above.

Another object of the present invention is to provide the use of a composition containing a Hovenia truncatula extract or its isolate in preparing a health functional food composition for preventing or improving coronavirus infectious diseases.

At this time, the descriptions of "Philipium orientalis extract", "its isolate", "coronavirus", "infectious disease", "prevention" and "improvement" are as described above.

Another object of the present invention is to provide the use of a composition containing Hovenia truncatula extract or its isolate in preparing feed compositions for preventing or improving coronavirus infectious diseases.

At this time, the descriptions of "Philipium orientalis extract", "its isolate", "coronavirus", "infectious disease", "prevention" and "improvement" are as described above.

Detailed ways

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are only used to illustrate the present invention, and the scope of the present invention is not limited to these.

Example 1: Preparation of Hovenia dulcis extract

To prepare the Hovenia dulcis extract, 1 L of water was added to 50 g, and hot water extraction was performed at 115° C. for 180 minutes using a heating plate (medical he ating plate; Gyeongseo Extractor Cosmos-600, Inchon, Korea), and a standard sample was used (standard testing sieves; 150 μm; Retsch, Haan, Germany) After filtration and freeze-drying, the samples were stored at -20°C until used in the experiments of the present invention. For use in experiments, it was dissolved in sterile distilled water at a concentration of 100 mg/ml, filtered through a 0.2 μm syringe filter, and stored at 4°C. During the experiment, 2% DMEM culture medium was used. Dilute it to the desired concentration and use it.

Example 2: Confirmation of inhibition of SARS-coronavirus-2 infection

Example 2-1: Preparing cell lines and viruses

In order to determine whether there is an inhibitory effect on the new SARS-coronavirus-2, Vero cells (verocells) were used. The Vero cells used in the present invention are American Type Culture Collection (ATCC, CCL-81; Manassas, VA) containing 10 % heat-inactivated fetal bovine serum and 1x Antibiotic-Antimycotic (Gibco/ThermoFisher Scientific, Waltham, MA) in Dulbecco's modified Eagle's medium (DMEM; Welgene, Gyeongsan, Korea) at 37°C under 5% carbon dioxide.

In addition, the new SARS-coronavirus-2 was provided and used by the Korea Centers for Disease Control and Prevention. Viral titers were measured using plaque assay.

Example 2-2: Confirmation that Hovenia dulcis extract inhibits SARS-coronavirus-2 infection

The Vero cell line (ATCC, CCL-81) was placed in a 96-well plate at a density of 2x10 ⁴ cells/well and cultured for one day to contain 100, 33, 11, 3.7 and 1.2 (μg/ ml) concentration, infect SARS-coronavirus-2 to 0.05MOI. Forty-eight hours after infection, cells were fixed with 1:3 acetone:methanol (v/v) at room temperature for 10 minutes, and then reacted with mouse primary antibody against coronavirus S (GeneT ex, Irvine, CA) protein. Virus-infected cells were stained using Alex Fluor 488-conjugated goat anti-mouse IgG (Invitrogen, Carlsbd, CA) as a secondary antibody. Since Alex Fluor 488 used as the secondary antibody emits green fluorescence, it can be observed Infected cells appear green. Cell nuclei were stained blue by DAPI staining. Using the drug Remdesivir as a control drug (standard drug), the effect of inhibiting SARS-coronavirus-2 infection was confirmed. Cell images were acquired using Operetta (PerkinElmer, Operetta) equipment, and infection rates were calculated using Harmony software. The case of virus infection alone was regarded as 100%, and the case of no infection was regarded as 0%, and the infection rate during drug treatment was calculated. The effective drug concentration (EC50) that inhibits viral infection by 50% was calculated using Prism software (Grap hPad, Prism). For cytotoxicity, cells were treated with only the same concentration and time of drugs in the absence of viral infection, and cell viability was measured by the MTS method. For cytotoxicity, cells were treated with only drugs at the same concentration for the same time in the absence of virus, and cell viability was measured by the MTS method. At this time, the concentration that reduced cell viability by 50% (CC50) was calculated as the cytotoxic concentration. The Selectivity Index (SI) is a measure of how good a compound is relative to its toxicity and is calculated by dividing the EC50 value by the CC50 value.

The specific results are shown in Figure 1.

The right vertical axis (Cell viability, black) represents the cytotoxicity (%) of the sample, and the left vertical axis (Antiviralactivity, red) represents the relative amount (%) of the sample inhibiting the S protein of SARS coronavirus-2. In addition, the specific experimental concentrations are 100, 33, 11, 3.7 and 1.2 (μg/ml).

As a result, as shown in Figure 1, the cytotoxicity (CC50) of the Hovenia dulcis extract of the present invention did not appear until the highest experimental concentration of 100 μg/ml, and the effective drug concentration (EC50) was 25.0 μg/ml, S.I. (selectivity) index, CC50/EC50) was determined to be >4. In addition, the CC50 of remdesivir in the positive control group was >100 μM, the EC50 value was 6.6 μM, and the SI was measured as >15.

It is thus confirmed that the Hovenia dulcis extract of the present invention has the effect of effectively inhibiting SARS-coronavirus-2 infection.

From the above description, those skilled in the art to which the present invention belongs will be able to understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the above-described embodiments are to be understood as illustrative and not restrictive in all respects. The scope of the present invention should be construed to include the meaning and scope of the following claims and all changes or modifications derived therefrom as compared with the above detailed description.

Claims (8)

1. An antiviral pharmaceutical composition against coronavirus containing an extract of Hovenia truncatula or its isolate. 2. The pharmaceutical composition according to claim 1, wherein the coronavirus is selected from the group consisting of SARS-coronavirus-2 (coronavirus-19), SARS-coronavirus and MERS-coronavirus. of at least one. 3. The pharmaceutical composition according to claim 1, characterized in that the Hoveicide extract is obtained by extracting Hovenia with water, C1-4 alcohol or a mixed solvent thereof. 4. The pharmaceutical composition according to claim 3, wherein the extraction is hot water extraction, ultrasonic extraction, room temperature extraction, cold soak extraction, reflux cooling extraction or steam extraction. 5. A pharmaceutical composition for preventing or treating coronavirus infectious diseases containing an extract of Hovenia truncatula or its isolate. 6. A method for preventing or treating coronavirus infectious diseases, comprising administering the pharmaceutical composition according to any one of claims 1 to 5 to non-human beings who have symptoms of coronavirus infection or are at risk of developing the disease. object steps. 7. A health functional food composition for preventing or improving coronavirus infectious diseases containing Hovenia truncatula extract or its isolate. 8. A feed composition for preventing or improving coronavirus infectious diseases containing an extract of Hovenia truncatula or its isolate.