Antiviral composition containing ginseng extract and steroid compound as active ingredients

Abstract

A composition containing a ginseng extract selected from the group consisting of ginseng extract, red ginseng extract, and black ginseng extract and a steroid compound as active ingredients has a broad range of virus-inhibiting activity and is therefore useful for effectively preventing, ameliorating, or treating viral infections or infectious diseases.

Description

[Technical Field]

[0001] The present invention relates to a composition containing, as active ingredients, at least one selected from the group consisting of Korean ginseng (including dry ginseng and undried ginseng), red ginseng, and black ginseng extracts, which are ginseng-related compounds having broad-spectrum antiviral activity against coronavirus, influenza, adenovirus, etc., and a steroid compound. [Background technology]

[0002] Virus is a Latin word meaning "toxic substance," and refers to a group of infectious, pathogenic particles that can pass through bacterial filter paper (0.22 μm). Viruses are found all around us, and some pathogenic microorganisms such as bacteria and fungi, as well as viruses, cause illness and produce foul odors, causing harm to the human body.

[0003] As a result, interest in hygiene management products that improve the environment against viruses and microorganisms is growing. For example, various products are being developed that can prevent infection with highly contagious viruses such as coronavirus disease 2019 (COVID-19), SARS, influenza virus, new strains of influenza, and Mers coronavirus (MERS-CoV).

[0004] Influenza viruses cause serious global diseases, claiming 250,000 to 500,000 lives worldwide each year, infecting 10 to 40% of children, and causing national economic losses.

[0005] Since the Spanish flu pandemic of 1918, influenza pandemics have occurred every few decades, such as the Asian flu of 1957 and the Hong Kong flu of 1968. The emergence of the 2009 H1N1 influenza virus (2009pdm) infected 270,000 people worldwide and killed more than 3,200. Despite pandemic preparedness measures being put in place due to the possibility of a highly contagious new mutant strain becoming rampant in the Southern Hemisphere, social repercussions and concerns have not abated.

[0006] Influenza is an acute respiratory illness accompanied by chills, fever, muscle aches, and coughing. It is typically transmitted from person to person through aerosols expelled into the air when an infected person coughs or sneezes. It can also be transmitted through bird feces and is often compared to the common cold. However, a common cold is a respiratory illness caused by infection with adenovirus, rhinovirus, coxsackievirus, or coronavirus, and typically does not accompany muscle aches or sudden high fever. Influenza infection is caused by influenza viruses belonging to the Osomyxoviridae family. As an RNA virus, antigenic variation is relatively easy, allowing repeated reinfection within an infected population.

[0007] Influenza viruses that infect humans are types A and B, and viral antigenic variation is determined by the HA (hemagglutinin) and NA (neuraminidase) on the viral surface. Genetic combinations of the viral surface proteins HA and NA produce subtypes H1-H16 and N1-N9, of which human influenza A virus subtypes are determined by three different combinations of H1, H2, and H3, and two different combinations of N1 and N2. Type B influenza cannot cause pandemics because, unlike type A, it only infects humans and does not inherit genes from animal viruses.

[0008] Adenoviruses (Adenoviridae) are medium-sized, non-enveloped viruses measuring 90-100 nm in size. They are icosahedral in shape and contain double-helical DNA. Adenoviruses are responsible for 5-10% of upper respiratory tract illnesses in children, and can also infect adults.

[0009] Unlike other viruses, which have a relatively mild clinical course compared to bacterial pneumonia, adenovirus can cause symptoms similar to those of severe bacterial pneumonia, leading to fatal outcomes and leaving sequelae such as permanent lung damage.

[0010] Viruses belonging to the Adenoviridae family can infect a variety of vertebrates, including humans, and were first isolated from the human adenoids, hence the name "adenovirus." The most common symptom of adenovirus infection is upper respiratory disease.

[0011] Adenovirus infections are often accompanied by conjunctivitis, tonsillitis, otitis media, laryngitis, gastroenteritis, etc. In children in particular, they can lead to bronchiolitis and pneumonia, and in rare cases, adenoviruses can also cause encephalitis and cystitis.

[0012] Coronaviruses are RNA viruses that cause respiratory, digestive, liver, and brain diseases in mammals and birds (Gallagher T.M. et al., Virology, 279(2):371-374, 2001). Among the coronaviruses, transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) are highly contagious viral diseases that invade the gastrointestinal system, causing vomiting, diarrhea, dehydration, and high fever. They also have high mortality rates and can result in significant economic losses (Duarte M. et al., J Gen Virol., 75(Pt 5):1195-1200, 1994).

[0013] Although these viruses have a very high mortality rate, the reality is that no reliable therapeutic agents have been developed for them, as with other viral infections.

[0014] Coronavirus disease 2019 (COVID-19), or corona-19, is a respiratory infection caused by a novel coronavirus (SARSCoV-2; an RNA virus belonging to the Coronaviridae family) that first emerged in Wuhan, China, in December 2019 and has since spread throughout China and the world.

[0015] Initially, it was known as a respiratory infection of unknown cause, but on January 9, 2020, the World Health Organization (WHO) announced that the cause of the pneumonia in question was a new coronavirus (SARS-CoV-2, named by the International Committee on Taxonomy of Viruses on February 11), confirming the pathogen.

[0016] The pathogen causing COVID-19 is SARS-CoV-2 (SARS-CoV-2). On February 11, 2020, the International Committee on Taxonomy of Viruses (ICTV) published a paper proposing the name SARS-CoV-2 for the pathogen causing COVID-19. The committee emphasized the virus's similarity to SARS (Severe Acute Respiratory Syndrome), which was prevalent in 2003.

[0017] South Korea obtained and analyzed the genetic base sequence of the virus, which China had released through academia, and found that it shared the highest homology (89.1%) with a similar coronavirus derived from bats. It also found low homology (39%-43%) with four human coronaviruses, 50% with MERS, and 77.5% with SARS.

[0018] Coronavirus disease 2019 (COVID-19), which is currently a global problem, is defined as a respiratory syndrome caused by infection with the new coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2). The disease classification is a legal infectious disease, Class 1 infectious disease, and the disease code is U07.1.

[0019] The pathogen is SARS-CoV-2, an RNA virus belonging to the Coronaviridae family, and so far it has been transmitted mainly through droplets (saliva) or contact, through droplets produced when coughing or sneezing, or by touching an object contaminated with the coronavirus and then touching one's eyes, nose, or mouth.

[0020] The global fatality rate for coronavirus infection is approximately 3.4% on average (WHO standard of 3.5). Currently, 6% of infected people worldwide are registered as deaths, with fatality rates varying greatly by country and age, with the elderly, those with weakened immune systems, and those with underlying diseases primarily experiencing severe illness and death.

[0021] Coronaviruses are RNA viruses with a single-positive strand helical structure and are composed of a total of five structural proteins, of which the spike glycoprotein (S protein) gives them the appearance of a crown or halo under an electron microscope (Schoeman and Fielding, 2019).Coronaviruses are the largest of all existing RNA viruses, measuring 27-34KB, and are subdivided into four groups: α, β, δ, and γ, of which the β group is highly infectious and virulent.

[0022] SARS-CoV-2 invades the host cell by binding to angiotensin converting enzyme 2 (ACE2) via the S protein present on its surface. ACE2 is distributed in large quantities in the oral and nasal mucosa, nasopharynx, lungs, stomach, small intestine, and large intestine, and is characterized by the observation of signs of viral pneumonia (Hamming et al., 2004).

[0023] The incubation period for coronavirus is 1 to 14 days (average 4 to 7 days), and the diagnostic criteria are to isolate the virus from a sample or to detect specific genes from a sample in accordance with diagnostic testing standards.

[0024] Under these circumstances, many efforts are being made to overcome the shortcomings of conventional antiviral preparations, and as part of these efforts, active research is being conducted in Korea on the antiviral efficacy of herbal extracts and plant extracts.

[0025] However, when applying the extract, most of the antiviral effects are low, and a solution to this is needed. Summary of the Invention [Problem to be solved by the invention]

[0026] Therefore, the problem to be solved by the present invention is to solve the above-mentioned problems and to provide a composition having excellent and broad-spectrum antiviral activity. [Means for solving the problem]

[0027] To solve the above problems, the present invention provides a composition for preventing, alleviating, or treating viral infections, which contains a ginseng extract selected from the group consisting of ginseng extract, red ginseng extract, and black ginseng extract, and a steroid compound as active ingredients.

[0028] In the composition according to the present invention, the ginseng extract is extracted using water or ethanol as an extraction solvent.

[0029] In the composition according to the present invention, the ginseng extract is extracted and then treated with at least one enzyme selected from the group consisting of β-galactosidase, β-glucosidase, β-glucanase, α-amylase, and cellulase.

[0030] In the composition according to the present invention, the ginseng extract is fermented with lactic acid bacteria or yeast.

[0031] In the composition according to the present invention, the ginseng extract is treated with an enzyme and then fermented with lactic acid bacteria or yeast.

[0032] In the composition according to the present invention, the steroid compound is at least one selected from the group consisting of dexamethasone, hydrocortisone, prednisolone, methylprednisolone, and triamcinolone.

[0033] In the composition according to the present invention, the virus may be any one of a coronavirus, an influenza virus, and an adenovirus. [Effects of the Invention]

[0034] The composition provided by the present invention has very significant inhibitory activity against a wide range of viruses, including coronavirus, influenza virus, and adenovirus, and can effectively prevent, ameliorate, or treat viral infections or diseases caused by infections.

[0035] In particular, from the perspective of suppressing viruses, the use of steroidal compounds can be disadvantageous. However, the composition of the present invention, even though it contains steroidal compounds, significantly increases antiviral effects such as the inhibition of viral proliferation, thereby solving the problems associated with the use of steroidal compounds and enabling the composition to fully utilize the beneficial aspects of steroidal compounds for people infected with viruses, such as the effect of alleviating inflammation such as pneumonia or bronchitis.

[0036] Therefore, the composition according to the present invention is a composition that is excellent in both anti-inflammatory effect that can alleviate symptoms caused by viral infection and antiviral effect that can inhibit viral proliferation. DETAILED DESCRIPTION OF THE INVENTION

[0037] Specific details for carrying out the present invention will be described below in detail.

[0038] The present invention relates to the discovery that a ginseng extract selected from the group consisting of ginseng extract, red ginseng extract, and black ginseng extract, an enzyme-treated product of such a ginseng extract, or a fermented product of such a ginseng extract, when used in combination with a steroid compound, exhibits a wide range of excellent antiviral activity, and the steroid compound also has an excellent anti-inflammatory effect against inflammation such as pneumonia and bronchitis, and is therefore effective as a composition that can prevent, ameliorate, or treat viral infections. Accordingly, the present invention provides a composition for preventing, ameliorating, or treating viral infections, which contains a ginseng extract, etc., and a steroid compound as active ingredients.

[0039] In the present invention described above, the steroid compound refers collectively to fat-soluble compounds having a stereonucleus, such as sterols, bile acids, and sex hormones, and is a lipid that does not contain fatty acids and has a common basic structure in which three rings of six carbon atoms and one ring of five carbon atoms, i.e., four rings, are bonded together. The steroid compound is an organic compound that is widely known in the technical field to which the present invention pertains, and is not particularly limited. Preferably, the steroid compound is at least one selected from the group consisting of dexamethasone, hydrocortisone, prednisolone, methylprednisolone, and triamcinolone.

[0040] These steroids have strong anti-inflammatory effects and play a major role in treating various conditions, including exacerbation of asthma and pulmonary diseases. They are effective in alleviating symptoms of viral infections such as bronchitis and pneumonia, preventing their aggravation, and treating them. However, they can sometimes weaken the immune system and delay the elimination of viruses. The present invention is designed to further enhance the antiviral effect of suppressing viral proliferation while maintaining the anti-inflammatory effect of the steroids when used in combination with a ginseng extract.

[0041] As described above, in the present invention, the steroid compounds are those currently approved as drugs and may be used in accordance with their standard amounts, preferably 1.5 to 10 mg of dexamethasone, 10 to 120 mg of hydrocortisone, 10 to 60 mg of prednisolone or methylprednisolone, and 8 to 16 mg of triamcinolone per 60 kg subject per day.

[0042] The ginseng extract used in the present invention is commonly referred to as ginseng extract (including dried ginseng or undried ginseng extract), red ginseng extract, and black ginseng extract. The methods for producing red ginseng and black ginseng are not particularly limited as long as they are widely known in the technical field to which the present invention pertains. In other words, ginseng, red ginseng, and black ginseng that are commonly used and distributed can be used without particular limitation.

[0043] Furthermore, the extraction method of the present invention can be any method that is widely known in the technical field to which the present invention pertains, and can be selected from, for example, a heat extraction method, a cold maceration extraction method, a reflux cooling extraction method, a steam distillation method, an ultrasonic extraction method, an elution method, a pressure method, and the like. Depending on the purpose, the extract may be subjected to a conventional fractionation step or purified by a conventional purification method.

[0044] In the present invention, it is preferable to use an extract obtained by a conventional extraction method using water or ethanol as an extraction solvent, or a mixture thereof, as the ginseng extract.

[0045] In addition, the primary extract can be powdered through additional processes such as vacuum distillation and freeze-drying or spray-drying, and purified fractions can be obtained through various types of chromatography such as silica gel column chromatography, high performance liquid chromatography, and thin layer chromatography.

[0046] Therefore, in the present invention, the extract can be understood as a concept including all extracts, separated compounds, fractions and purified products obtained at each stage of extraction, fractionation or purification, as well as diluted, concentrated and dried products thereof.

[0047] In the present invention, the ginseng extract may be used after being treated with an enzyme or fermented, or after being treated with an enzyme and then fermented.

[0048] The enzyme used in the present invention is preferably at least one selected from the group consisting of β-galactosidase, β-glucosidase, β-glucanase, α-amylase, and cellulase.

[0049] The strain used for the fermentation of the present invention may be any strain known to ferment ginseng extracts, and preferably may be a lactic acid bacterium or yeast.

[0050] Specifically, the ginseng extract used in the present invention can be produced by the following steps: an extraction step of extracting at least one ginseng selected from the group consisting of ginseng (dried ginseng or unripe ginseng), red ginseng and black ginseng with hot water at 70 to 100°C, or an extraction step of extracting ginseng with 70% by weight ethanol; an enzyme treatment step in which an enzyme is added to the extract of the extraction step and the extract is treated with the enzyme at 25 to 65°C for 2 to 7 hours; The extract after the enzyme treatment may be subjected to a fermentation step in which lactic acid bacteria or yeast, preferably Saccharomyces cerevisiae, is added and fermented at 20 to 40° C. for 10 to 48 hours.

[0051] The amount of the ginseng extract used is not particularly limited, but is preferably 2 to 5 g per 60 kg of subject per day on a solid basis.

[0052] The composition according to the present invention, which comprises the black ginseng extract and the steroid compound, may be administered once a day at the above-mentioned daily standard dosage, but it is preferable to administer it in two or three divided doses.

[0053] The composition according to the present invention is believed to exhibit antiviral activity without particular limitations because it increases the mRNA expression level of Ifnb1 (interferon beta), a type 1 interferon that exhibits broad antiviral activity, and the mRNA expression levels of Mx1 and Mx2, genes that express Mx1 (MX Dynamin Like GTPase 1) and Mx2 (MX Dynamin Like GTPase 2). Examples of such antiviral activity include those against viruses of the Flaviviridae, Adenovirinae, Coronavirinae, Reoviridae, Picornaviridae, Caliciviridae, Togaviridae, Arenaviridae, and other families. Examples of such viruses include the renaviridae, Orthomyxoviridae, Paramyxoviridae, Bunyaviridae, Rhabdoviridae, Filoviridae, Astroviridae, Bornaviridae, and Arteriviridae, and preferably, the antiviral agent exhibits antiviral activity against coronaviruses, influenza viruses, and adenoviruses.

[0054] In particular, in the case of the coronaviruses mentioned above, it shows excellent antiviral activity against all of HCoV-229E, HCoV-NL63, SARS-CoV, MERS-CoV, HCoV-OC43, HCoV-HKU1, and SARS-CoV-2.

[0055] The composition of the present invention described above may be used as a pharmaceutical composition, a food composition, a food additive composition, a feed composition, or a feed additive composition. The additives, excipients, other antiviral agents, etc. used in producing these compositions may be used in accordance with normal procedures without any particular limitations, and the formulation of the composition is also not particularly limited.

[0056] The present invention will be described in more detail with reference to the following examples and test examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of explaining the present invention in more detail, and that the scope of the present invention is not limited by these examples according to the gist of the present invention.

[0057] Examples of ginseng use Preparation of the composition The ginseng extracts used were a hot water extract, an enzyme-treated product obtained by enzymatically treating the extract with α-amylase, and a fermented product obtained by fermenting the extract with Saccharomyces cerevisiae.

[0058] specifically, Extraction step of extracting undried ginseng with hot water at 70-100℃; an enzyme treatment step in which an enzyme is added to the extract of the extraction step and the extract is treated with the enzyme at 45°C for 3 hours; or The extract from the extraction step was added to Saccharomyces cerevisiae and fermented at 30°C for 24 hours to produce the product.

[0059] Each ginseng extract was combined with any one selected from the group consisting of dexamethasone, hydrocortisone, prednisolone, and triamcinolone to prepare a composition according to the present invention. A composition using a ginseng extract and a steroid compound alone was used as a comparative example, and its composition is shown in Table 1 below.

[0060] [Table 1]

[0061] Test Example 1: Mouse preparation The inventors of the present invention purchased 8-week-old C57BL / 6 male mice from the Central Laboratory Animal Center and used them in the following experiments. They were acclimatized for one week in a laboratory environment with a temperature of 21±2°C, humidity of 50±10%, and a 12 / 12 hour dark / light cycle, with sufficient food and water provided.

[0062] Next, 8-week-old C57BL / 6 mice were orally administered a combination of 22.5 mg / kg solid ginseng extract, 0.03 mg / kg dexamethasone, 2.5 mg / kg hydrocortisone, 0.1 mg / kg prednisolone, and 0.1 mg / kg triamcinolone twice daily for 2 weeks, and then sacrificed to remove the livers, which were used in the following experiments.

[0063] Confirmation of antiviral effect To confirm the antiviral effects of the compositions prepared in the Examples and Comparative Examples, livers excised from mice treated with the compositions and from a control group were pulverized using a Tissue Lyser II (Qiagen) and total RNA was isolated using an RNeasy mini kit (Qiagen). Reverse transcription (RT) was then performed using 2 μg of RNA with a RevertAid RT Kit (EP0441, Thermo Fisher Scientific), followed by real-time qRT-PCR using a SensiFAST SYBR Hi-ROX kit (BIO92020, Bioline, London, UK).

[0064] The qPCR results were obtained by quantifying the target gene through the housekeeping gene Gapdh. Table 2 shows the mRNA expression levels of Ifnb1 (interferon beta), a type 1 interferon, and the mRNA expression levels of Mx1 and Mx2, which express Mx1 (MX Dynamin Like GTPase 1) and Mx2 (MX Dynamin Like GTPase 2), proteins that exhibit broad-spectrum antiviral effects. The values ​​in Table 2 represent relative expression levels, with the expression level of the untreated control group set at 1.

[0065] [Table 2]

[0066] As shown in Table 2, both the composition of the present invention and the comparative composition containing ginseng extract alone increased the mRNA expression levels of Ifnb1 (interferon beta), a type 1 interferon, and the mRNA expression levels of Mx1 and Mx2, genes that express Mx1 (MX Dynamin Like GTPase 1) and Mx2 (MX Dynamin Like GTPase 2), proteins that exhibit broad-spectrum antiviral effects. However, the extent of the increase was more pronounced in the composition of the present invention than in the comparative composition.

[0067] Furthermore, in Comparative Examples 4 to 7, in which steroid compounds were used, the use of steroid compounds may be disadvantageous from the viewpoint of suppressing the virus by reducing it. However, the composition of the present invention, even though it contains steroid compounds, exhibits a significantly increased antiviral effect, solving the problems associated with the use of steroid compounds, and is effective in alleviating inflammation such as pneumonia and bronchitis, making it possible to fully utilize its useful aspects for people infected with viruses.

[0068] Furthermore, when comparing examples according to the present invention, the examples in which ginseng extract was subjected to enzyme treatment or fermentation treatment showed an improved increase in the mRNA expression level of Ifnb1 (interferon beta), a type 1 interferon, and the mRNA expression levels of Mx1 and Mx2, genes that express Mx1 (MX Dynamin Like GTPase 1) and Mx2 (MX Dynamin Like GTPase 2), proteins that exhibit broad-spectrum antiviral effects.

[0069] Type I interferons (IFNs) are proteins secreted primarily by virus-infected cells. The most common type I interferons in humans are IFN-α and IFN-β, which exhibit a broad range of antiviral effects (Nat Rev Immunol. 2015 Feb;15(2):87-103.).

[0070] In addition, the Mx1 protein (Interferon-induced GTP-binding protein Mx1) and the Mx2 protein (Interferon-induced GTP-binding protein MX2) are both induced by type 1 interferon, and the interferon-induced Mx protein is a marker that accumulates in the cytoplasm in humans and is known to bind to invading viruses and induce their death (Trends Microbiol. 2015 Mar; 23(3): 154-63., Microbiol Mol Biol Rev. 2013 Dec; 77(4): 551-66.).

[0071] Therefore, the above results indicate that when the composition according to the present invention is administered during viral infection, an antiviral effect is observed, and that it can be used to effectively prevent, ameliorate, or treat viral infections or diseases caused by infections.

[0072] Test example 2: Confirmation of antiviral effect through in vitro experiments Vero-E6 cells were cultured in DMEM(- / -) and 5 × 10 cells were placed in a 12-well plate for virus infection experiments. 5 The cells were seeded at a concentration of 1000 cells / well and cultured at 37°C in a CO2 incubator for 24 hours. After removing the cell culture medium, the cells were washed twice with 1x PBS.

[0073] The cells were pretreated with the composition of Test Example 1 at a concentration of 100 μg / ml and then cultured in a CO2 incubator at 37°C for 2 hours. After that, the cell culture medium was removed for viral infection, and the cells were treated with the virus shown in Table 7 at a concentration of 50 pfu / well for 1 hour. After that, the cell culture medium was removed, and 1.5 ml of medium was added to each well. After incubation for 72 hours, the results were confirmed and are shown in Table 3. The values ​​in Table 3 are relative viral titers, with the viral titer of the untreated control group being set at 100.

[0074] [Table 3]

[0075] As shown in the test results in Table 3, both the composition of the example and the comparative example using ginseng extract alone reduced viral RNA, but compared to the comparative example, the composition of the present invention showed a reduction of more than 70%, indicating significantly higher antiviral activity.

[0076] Furthermore, in Comparative Examples 4 to 7, in which steroid compounds were used, the viral RNA of the virus increased, which had the opposite effect in terms of inhibiting viral proliferation. However, when used in combination with ginseng extract as in the present invention, the antiviral activity of the ginseng extract is increased, thereby solving the problems seen in Comparative Examples 4 to 7. The composition of the present invention is a very useful composition that has strong antiviral activity and can fully utilize the anti-inflammatory effect of steroid compounds.

[0077] Furthermore, when comparing the examples according to the present invention, the reduction in viral RNA was improved in the examples in which the ginseng extract was subjected to enzyme treatment or fermentation treatment.

[0078] Test example 3: Confirmation of antiviral effect through in vivo experiments To confirm the antiviral effect in vivo, 9-week-old K18-ACE2 TG mice were used in the experiment. They were acclimatized for one week in a laboratory environment with a temperature of 21±2°C, humidity of 50±10%, and a 12 / 12 hour dark / light cycle, with sufficient food and water. They were then orally administered the drug once, as in Test Example 1, one day before viral infection.

[0079] Three days after virus infection, four mice were orally administered and sacrificed to obtain lung tissue. The virus titers in the lung tissue were measured by plaque assay and are shown in Table 4. The values ​​in Table 4 are relative virus titers, with the virus titer in the untreated control group set at 100.

[0080] [Table 4]

[0081] As shown in the test results in Table 4, unlike the comparative example using a steroid compound, the viral titer was reduced in both the composition of the example according to the present invention and the comparative example using ginseng extract. However, when compared to the comparative example using ginseng extract, the composition according to the present invention showed a reduction of more than 70%, indicating significantly higher antiviral activity.

[0082] Furthermore, when comparing the examples of the present invention, the extent of reduction in viral titer was improved in the examples in which the ginseng extract was subjected to enzyme treatment or fermentation treatment.

[0083] Examples of red ginseng use Preparation of the composition The examples were the same as those using ginseng, except that red ginseng extract was used. The comparative examples using a steroid compound alone were omitted because the test results were similar to those of the examples using ginseng. The structures of the examples and comparative examples are shown in Table 5 below.

[0084] [Table 5]

[0085] Test Example 1: Mouse preparation The inventors of the present invention purchased 8-week-old C57BL / 6 male mice from the Central Laboratory Animal Center and used them in the following experiments. They were acclimatized for one week in a laboratory environment with a temperature of 21±2°C, humidity of 50±10%, and a 12 / 12 hour dark / light cycle, with sufficient food and water provided.

[0086] Next, 8-week-old C57BL / 6 mice were orally administered a combination of 22.5 mg / kg solid red ginseng extract, 0.03 mg / kg dexamethasone, 2.5 mg / kg hydrocortisone, 0.1 mg / kg prednisolone, and 0.1 mg / kg triamcinolone twice daily for 2 weeks, and then sacrificed to remove the livers, which were used in the following experiments.

[0087] Confirmation of antiviral effect To confirm the antiviral effects of the compositions prepared in the Examples and Comparative Examples, livers excised from mice treated with the compositions and from a control group were pulverized using a Tissue Lyser II (Qiagen) and total RNA was isolated using an RNeasy mini kit (Qiagen). Reverse transcription (RT) was then performed using 2 μg of RNA with a RevertAid RT Kit (EP0441, Thermo Fisher Scientific), followed by real-time qRT-PCR using a SensiFAST SYBR Hi-ROX kit (BIO92020, Bioline, London, UK).

[0088] The qPCR results were obtained by quantifying the target gene through the housekeeping gene Gapdh. Table 6 shows the mRNA expression levels of Ifnb1 (interferon beta), a type 1 interferon, and the mRNA expression levels of Mx1 and Mx2, which express Mx1 (MX Dynamin Like GTPase 1) and Mx2 (MX Dynamin Like GTPase 2), proteins that exhibit broad-spectrum antiviral effects. The values ​​in Table 6 represent relative expression levels, with the expression level of the control group, which received no treatment, set at 1.

[0089] [Table 6]

[0090] As shown in Table 6, both the composition of the present invention and the comparative composition using red ginseng extract alone increased the mRNA expression levels of Ifnb1 (interferon beta), a type 1 interferon, and Mx1 and Mx2, which express Mx1 (MX Dynamin Like GTPase 1) and Mx2 (MX Dynamin Like GTPase 2), proteins that exhibit broad-spectrum antiviral activity. However, the degree of increase was more pronounced in the composition of the present invention than in the comparative composition. When compared with the examples of the present invention, the degree of increase was even greater in the examples in which the red ginseng extract was subjected to enzyme treatment or fermentation treatment.

[0091] Furthermore, compared to the example using Korean ginseng, in this example using red ginseng, the mRNA expression levels of Ifnb1 (interferon beta), a type 1 interferon, and the mRNA expression levels of Mx1 and Mx2, genes that express Mx1 (MX Dynamin Like GTPase 1) and Mx2 (MX Dynamin Like GTPase 2), proteins that exhibit broad-spectrum antiviral effects, were generally increased, indicating that red ginseng is superior to Korean ginseng in terms of antiviral activity.

[0092] Type I interferons (IFNs) are proteins secreted primarily by virus-infected cells. The most common type I interferons in humans are IFN-α and IFN-β, which exhibit a broad range of antiviral effects (Nat Rev Immunol. 2015 Feb;15(2):87-103.).

[0093] In addition, the Mx1 protein (Interferon-induced GTP-binding protein Mx1) and the Mx2 protein (Interferon-induced GTP-binding protein MX2) are both induced by type 1 interferon, and the interferon-induced Mx protein is a marker that accumulates in the cytoplasm in humans and is known to bind to invading viruses and induce their death (Trends Microbiol. 2015 Mar; 23(3): 154-63., Microbiol Mol Biol Rev. 2013 Dec; 77(4): 551-66.).

[0094] Therefore, the above results indicate that when the composition according to the present invention is administered during viral infection, an antiviral effect is observed, and that it can be used to effectively prevent, ameliorate, or treat viral infections or diseases caused by infections.

[0095] Test example 2: Confirmation of antiviral effect through in vitro experiments Vero-E6 cells were cultured in DMEM(- / -) and 5 × 10 cells were placed in a 12-well plate for virus infection experiments. 5 The cells were seeded at a concentration of 1000 cells / well and cultured at 37°C in a CO2 incubator for 24 hours. After removing the cell culture medium, the cells were washed twice with 1x PBS.

[0096] The cells were pretreated with the composition of Test Example 1 at a concentration of 100 μg / ml and then cultured in a CO2 incubator at 37°C for 2 hours. After that, the cell culture medium was removed for viral infection, and the cells were treated with the virus shown in Table 7 at a concentration of 50 pfu / well for 1 hour. After that, the cell culture medium was removed, and 1.5 ml of medium was added to each well. After incubation for 72 hours, the results were confirmed and are shown in Table 7. The values ​​in Table 7 are relative viral titers, with the viral titer of the untreated control group being set at 100.

[0097] [Table 7]

[0098] As shown in Table 7, the viral RNA of the virus was reduced in both the Example and the Comparative Example in which red ginseng extract was used alone. However, compared to the Comparative Example, the composition according to the present invention showed a reduction of more than 70%, indicating that it has significantly higher antiviral activity. Compared to the Examples according to the present invention, the extent of viral RNA reduction was improved in the Examples in which red ginseng extract was subjected to enzyme treatment or fermentation treatment.

[0099] In addition, compared to the example using Korean ginseng, the example using red ginseng showed an overall improvement in the reduction of viral RNA, which means that red ginseng is superior to Korean ginseng in terms of antiviral activity.

[0100] Test example 3: Confirmation of antiviral effect through in vivo experiments To confirm the antiviral effect in vivo, 9-week-old K18-ACE2 TG mice were used in the experiment. They were acclimatized for one week in a laboratory environment with a temperature of 21±2°C, humidity of 50±10%, and a 12 / 12 hour dark / light cycle, with sufficient food and water. They were then orally administered the drug once, as in Test Example 1, one day before viral infection.

[0101] Three days after virus infection, four mice were orally administered and sacrificed to obtain lung tissue. The virus titers in the lung tissue were measured using plaque assay, and the results are shown in Table 8. The values ​​in Table 8 are relative virus titers, with the virus titer in the untreated control group set at 100.

[0102] [Table 8]

[0103] As shown in Table 8, the viral titer was reduced in both the composition of the example according to the present invention and the comparative example using red ginseng extract. However, the degree of reduction was significantly improved in the composition of the present invention compared to the comparative example using red ginseng extract. When compared to the examples according to the present invention, the degree of reduction of viral titer was improved in the examples in which red ginseng extract was subjected to enzyme treatment or fermentation treatment.

[0104] In addition, compared to the example using Korean ginseng, the example using red ginseng showed an overall improvement in the reduction of viral titers, which means that red ginseng is superior to Korean ginseng in terms of antiviral activity.

[0105] Examples of black ginseng use Preparation of the composition The examples are the same as those using ginseng, except that black ginseng extract was used. Also, the comparative examples using a steroid compound alone have the same test results as those in the examples using ginseng, so they are omitted. The structures of the examples and comparative examples are shown in Table 9 below.

[0106] [Table 9]

[0107] Test Example 1: Mouse preparation The inventors of the present invention purchased 8-week-old C57BL / 6 male mice from the Central Laboratory Animal Center and used them in the following experiments. They were acclimatized for one week in a laboratory environment with a temperature of 21±2°C, humidity of 50±10%, and a 12 / 12 hour dark / light cycle, with sufficient food and water provided.

[0108] Next, 8-week-old C57BL / 6 mice were orally administered a combination of 22.5 mg / kg of solid black ginseng extract, 0.03 mg / kg dexamethasone, 2.5 mg / kg hydrocortisone, 0.1 mg / kg prednisolone, and 0.1 mg / kg triamcinolone twice daily for 2 weeks, and then sacrificed to remove the livers, which were used in the following experiments.

[0109] Confirmation of antiviral effect To confirm the antiviral effects of the compositions prepared in the Examples and Comparative Examples, livers excised from mice treated with the compositions and from a control group were pulverized using a Tissue Lyser II (Qiagen) and total RNA was isolated using an RNeasy mini kit (Qiagen). Reverse transcription (RT) was then performed using 2 μg of RNA with a RevertAid RT Kit (EP0441, Thermo Fisher Scientific), followed by real-time qRT-PCR using a SensiFAST SYBR Hi-ROX kit (BIO92020, Bioline, London, UK).

[0110] The qPCR results were obtained by quantifying the target gene through the housekeeping gene Gapdh. Table 10 shows the mRNA expression levels of Ifnb1 (interferon beta), a type 1 interferon, and the mRNA expression levels of Mx1 and Mx2, which express Mx1 (MX Dynamin Like GTPase 1) and Mx2 (MX Dynamin Like GTPase 2), proteins that exhibit broad-spectrum antiviral effects. The values ​​in Table 10 represent relative expression levels, with the expression level of the control group, which received no treatment, set at 1.

[0111] [Table 10]

[0112] As shown in Table 10, both the composition according to the present invention and the comparative composition using black ginseng extract alone increased the mRNA expression levels of Ifnb1 (interferon beta), a type 1 interferon, and Mx1 and Mx2, which express Mx1 (MX Dynamin Like GTPase 1) and Mx2 (MX Dynamin Like GTPase 2), proteins that exhibit broad-spectrum antiviral activity. However, the degree of increase was more pronounced in the composition according to the present invention than in the comparative composition. Compared with the examples according to the present invention, the degree of increase was even greater in the examples in which the black ginseng extract was subjected to enzyme treatment or fermentation treatment.

[0113] Furthermore, compared with the examples using Korean ginseng and red ginseng, the examples using black ginseng showed an overall increase in the mRNA expression levels of Ifnb1 (interferon beta), a type 1 interferon, and the mRNA expression levels of Mx1 and Mx2, genes that express Mx1 (MX Dynamin Like GTPase 1) and Mx2 (MX Dynamin Like GTPase 2), proteins that exhibit broad-spectrum antiviral effects, indicating that black ginseng is superior to Korean ginseng in terms of antiviral activity.

[0114] Type I interferons (IFNs) are proteins secreted primarily by virus-infected cells. The most common type I interferons in humans are IFN-α and IFN-β, which exhibit a broad range of antiviral effects (Nat Rev Immunol. 2015 Feb;15(2):87-103.).

[0115] In addition, the Mx1 protein (Interferon-induced GTP-binding protein Mx1) and the Mx2 protein (Interferon-induced GTP-binding protein MX2) are both induced by type 1 interferon, and the interferon-induced Mx protein is a marker that accumulates in the cytoplasm in humans and is known to bind to invading viruses and induce their death (Trends Microbiol. 2015 Mar; 23(3): 154-63., Microbiol Mol Biol Rev. 2013 Dec; 77(4): 551-66.).

[0116] Therefore, the above results indicate that when the composition according to the present invention is administered during viral infection, an antiviral effect is observed, and that it can be used to effectively prevent, ameliorate, or treat viral infections or diseases caused by infections.

[0117] Test example 2: Confirmation of antiviral effect through in vitro experiments Vero-E6 cells were cultured in DMEM(- / -) and 5 × 10 cells were placed in a 12-well plate for virus infection experiments. 5 The cells were seeded at a concentration of 1000 cells / well and cultured at 37°C in a CO2 incubator for 24 hours. After removing the cell culture medium, the cells were washed twice with 1x PBS.

[0118] After pre-treating with the composition of Test Example 1 at a concentration of 100 μg / ml, the cells were cultured at 37°C in a CO2 incubator for 2 hours. After that, the cell culture medium was removed for viral infection, and the cells were treated with the virus shown in Table 11 at a concentration of 50 pfu / well for 1 hour. After that, the cell culture medium was removed, and 1.5 ml of medium was added to each well. After incubation for 72 hours, the results were confirmed and are shown in Table 11. The values ​​in Table 11 are relative viral titers, with the viral titer of the untreated control group being set at 100.

[0119] [Table 11]

[0120] The test results in Table 11 show that viral RNA was reduced in both the Example and the Comparative Example in which black ginseng extract was used alone. However, when compared with the Comparative Example, the composition according to the present invention showed significantly higher antiviral activity. When compared with the Example according to the present invention, the Example in which black ginseng extract was subjected to enzyme treatment or fermentation treatment showed a higher degree of reduction in viral RNA.

[0121] In addition, compared to the examples using Korean ginseng and red ginseng, the example using black ginseng showed an overall improvement in the reduction of viral RNA, which means that black ginseng is superior to Korean ginseng and red ginseng in terms of antiviral activity.

[0122] Test example 3: Confirmation of antiviral effect through in vivo experiments To confirm the antiviral effect in vivo, 9-week-old K18-ACE2 TG mice were used in the experiment. They were acclimatized for one week in a laboratory environment with a temperature of 21±2°C, humidity of 50±10%, and a 12 / 12 hour dark / light cycle, with sufficient food and water. They were then orally administered the drug once, as in Test Example 1, one day before viral infection.

[0123] Three days after virus infection, four mice were orally administered and sacrificed to obtain lung tissue. The virus titers in the lung tissue were measured using plaque assay, and the results are shown in Table 12. The values ​​in Table 12 are relative virus titers, with the virus titer in the untreated control group set at 100.

[0124] [Table 12]

[0125] As shown in Table 12, the viral titer was reduced in both the composition of the example according to the present invention and the comparative example using black ginseng extract. However, the degree of reduction was significantly improved in the composition of the present invention compared to the comparative example using black ginseng extract. When compared to the examples according to the present invention, the degree of reduction of viral titer was improved in the examples in which black ginseng extract was subjected to enzyme treatment or fermentation treatment.

[0126] In addition, compared to the examples using Korean ginseng and red ginseng, the example using black ginseng showed an overall improvement in the reduction of viral titers, which means that black ginseng is superior to Korean ginseng in terms of antiviral activity.

[0127] In summary, it has been found that the composition according to the present invention can be used as a therapeutic agent for various viral infections or infectious diseases.

[0128] Therefore, the composition provided by the present invention has very significant inhibitory activity against a wide range of viruses, including coronavirus, influenza virus, and adenovirus, and can effectively prevent, ameliorate, or treat viral infections or diseases caused by infections.

[0129] Furthermore, the present invention has the advantage of avoiding side effects such as weakening of the immune system that may be caused by steroid compounds by using a steroid compound in combination with a ginseng extract.

[0130] Therefore, the composition according to the present invention is an excellent composition in terms of both the anti-inflammatory effect that can alleviate symptoms caused by viral infection and the antiviral effect that can inhibit viral proliferation.

Claims

1. The product contains a ginseng extract selected from the group consisting of ginseng extract, red ginseng extract, and black ginseng extract, and a steroid compound as active ingredients. A pharmaceutical composition for preventing, alleviating or treating a viral infection, comprising:

2. The product contains a ginseng extract selected from the group consisting of ginseng extract, red ginseng extract, and black ginseng extract, and a steroid compound as active ingredients. A health food composition for preventing or ameliorating viral infections, comprising:

3. The product contains a ginseng extract selected from the group consisting of ginseng extract, red ginseng extract, and black ginseng extract, and a steroid compound as active ingredients. A feed composition for preventing or ameliorating viral infections, comprising:

4. The virus is any one of a coronavirus, an influenza virus, and an adenovirus. The composition according to any one of claims 1 to 3.

5. The ginseng extract was extracted using water or ethanol as an extraction solvent. The composition according to any one of claims 1 to 3.

6. After extraction, the ginseng extract was treated with at least one enzyme selected from the group consisting of β-galactosidase, β-glucosidase, β-glucanase, α-amylase, and cellulase. The composition of claim 5.

7. The ginseng extract is extracted and then fermented with lactic acid bacteria or yeast. The composition of claim 5.

8. The ginseng extract is treated with an enzyme and then fermented with lactic acid bacteria or yeast. The composition of claim 6.

9. The steroid compound is at least one selected from the group consisting of dexamethasone, hydrocortisone, prednisolone, methylprednisolone, and triamcinolone. The composition according to any one of claims 1 to 3.

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