Antiviral composition comprising ginseng-based extract and steroid-based compound as active ingredients

Abstract

The composition comprising a ginseng-based extract selected from the group consisting of a ginseng extract, a red ginseng extract, and a black ginseng extract and a steroid compound as active ingredients according to the present invention has a broad virus inhibitory activity, and thus can be used for effectively preventing, improving, or treating infections caused by viruses or diseases caused by infections.

Description

Technical Field

[0001] The present invention relates to a composition comprising one or more selected from the group consisting of extracts of ginseng (including dried ginseng, fresh ginseng), red ginseng extract, and black ginseng extract and steroid compounds as active ingredients. These ginsengs can be considered to have broad antiviral activities against coronavirus, influenza virus, adenovirus, etc. Background Art

[0002] Virus means a toxic substance in Latin and refers to a group of infectious pathogenic particles that can pass through a bacterial filter paper (0.22 μm). Viruses exist around us. Some pathogenic microorganisms such as bacteria and molds, and viruses can cause diseases and produce bad odors, causing harm to the human body.

[0003] Therefore, there is an increasing interest in hygiene management products aimed at improving the living environment of these viruses and microorganisms. For example, various products are being developed to prevent highly prevalent virus infections themselves, such as SARS, including the novel coronavirus 2019 (COVID-19), influenza virus, novel influenza, Middle East respiratory syndrome coronavirus (MERS-CoV), etc.

[0004] Influenza virus causes 250,000 to 500,000 deaths globally every year, infects 10% to 40% of children, and triggers serious global diseases, causing economic losses to countries.

[0005] Influenza is an acute respiratory disease accompanied by chills, fever, muscle pain, and cough. It is usually transmitted from person to person through aerosols released into the air by infected people coughing or sneezing, causing infection. It can also be transmitted through the excrement of birds, etc. Compared with the common cold, it is a respiratory disease caused by infections with adenovirus, rhinovirus, coxsackievirus, coronavirus, etc., and usually does not involve muscle pain or a rapid high fever. Influenza infection is caused by the influenza virus belonging to the family Orthomyxoviridae. As an RNA virus, antigenic variation is relatively easy, so repeated infections may occur in the infected population.

[0006] There are two types of influenza viruses that cause human infection: type A and type B. The antigenic variation of the virus is determined by the hemagglutinin (HA) and neuraminidase (NA) on the surface of the virus. The H1-H16 and N1-N9 subtypes are generated based on the genetic combination of the virus surface proteins HA and NA. Among them, the subtype of type A human influenza virus is determined by different combinations of three types: H1, H2 and H3, and two types: N1 and N2. The reason why type B influenza cannot cause a pandemic is that, unlike type A, it only infects humans and does not obtain genes from animal viruses.

[0007] Adenovirus (Adenoviridae) is a medium-sized virus, 90 to 100 nm in size, without an envelope, in the shape of a regular icosahedron, with double-helical DNA. Adenovirus is the cause of 5 to 10% of upper respiratory tract diseases in children, and adults are also infected.

[0008] Unlike other viruses, whose clinical course is relatively benign compared to bacterial pneumonia, adenoviruses can present with symptoms similar to severe bacterial pneumonia and are known to cause fatal outcomes or leave sequelae such as permanent lung damage.

[0009] Viruses belonging to the Adenoviridae family can infect a variety of vertebrates, including humans. Adenoviruses were originally isolated from human tonsils (adenoids), hence the name "adenovirus". The most common symptom of adenovirus infection is upper respiratory tract disease.

[0010] Adenovirus infection is often accompanied by conjunctivitis, tonsillitis, otitis media, laryngitis, gastroenteritis, etc. Children in particular may suffer from bronchiolitis or pneumonia. In rare cases, adenovirus can also cause encephalitis or cystitis.

[0011] Coronavirus is an RNA virus that can cause respiratory, digestive, liver, and brain diseases in mammals and birds (Gallagher TM et al., Virology, 279(2):371 - 374, 2001), etc. Among the viruses belonging to the coronavirus, in particular, transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) are viruses that cause highly contagious viral diseases. They invade the gastrointestinal and digestive systems, leading to dehydration and high fever caused by vomiting and diarrhea, and have a very high fatality rate, causing significant economic losses (Duarte M et al., J Gen Virol., 75(Pt5):1195 - 1200, 1994).

[0012] Although the fatality rates of these viruses are very high, no definite therapeutic agents have been developed, unlike diseases caused by other viral infections.

[0013] Coronavirus disease 2019 (COVID - 19), or simply COVID - 19, is a respiratory infection disease caused by the novel coronavirus (SARS - CoV - 2; an RNA virus belonging to the family Coronaviridae).

[0014] It was initially only considered an unexplained respiratory infectious disease. However, on January 9, 2020, the World Health Organization (WHO) announced that the pathogen of the pneumonia was the novel coronavirus (SARS - CoV - 2, named by the International Committee on Taxonomy of Viruses on February 11), and the pathogen was confirmed.

[0015] The pathogen of coronavirus disease 2019 is "SARS - CoV - 2". The International Committee on Taxonomy of Viruses (ICTV) published a paper on February 11, 2020, recommending naming the pathogen of COVID - 19 as SARS - CoV - 2. The committee stated that it emphasized the similarity of this virus to the SARS (severe acute respiratory syndrome) that prevailed in 2003.

[0016] South Korea obtained and analyzed the gene base sequence of the virus released by China through the academic community, and confirmed that its homology with a similar coronavirus of bat origin is the highest (89.1%). Its homology with four human coronaviruses is relatively low, at 39% - 43%, while its homology with MERS is 50% and with SARS is 77.5%.

[0017] The disease of Coronavirus disease 2019 (COVID-19) is a current global challenge. It is defined as a respiratory syndrome caused by the novel coronavirus (Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2). The disease is classified as a legally notifiable infectious disease, a class I infectious disease, and a newly emerging infectious disease syndrome, with the disease code U07.1.

[0018] The pathogen is SARS-CoV-2, an RNA virus belonging to the family Coronaviridae. Currently, the main routes of transmission are through droplets (saliva droplets) or contact, either by droplets generated during coughing or sneezing, or by touching the eyes, nose, or mouth after touching an object contaminated with the coronavirus.

[0019] The global mortality rate of coronavirus infection is approximately 3.4% (WHO, 3.5 standard). Currently, the global mortality rate among infected individuals is 6%. The case fatality rate varies significantly by country and age. Severe cases and deaths mainly occur in elderly patients, patients with immunosuppression, and patients with underlying diseases.

[0020] Coronaviruses are RNA viruses with a single positive strand helical structure, consisting of a total of five structural proteins. Due to the presence of spike glycoprotein (S protein), they appear to have a crown or halo under electron microscopy (Schoeman and Fielding, 2019). Coronaviruses are 27 - 34 KB in size, the largest among existing RNA viruses, and are divided into four groups: α, β, δ, and γ, with the β group being highly infectious and virulent.

[0021] The S protein (S protein) present on the surface of SARS-CoV-2 binds to the angiotensin converting enzyme 2 (ACE2) of the host cell and invades. ACE2 is widely distributed in the oral cavity, nasal mucosa, nasopharynx, lung, stomach, small intestine, large intestine, etc., and is characterized by viral pneumonia (Hamming et al., 2004).

[0022] The incubation period of the coronavirus is 1 - 14 days (average 4 - 7 days). As a diagnostic criterion, the diagnosis is made by isolating the virus from the sample or detecting specific genes in the sample according to the diagnostic testing criteria.

[0023] In this case, many efforts are being made to overcome the drawbacks of existing antiviral agents. As one of these efforts, South Korea is actively conducting research on the antiviral efficacy of herbal extracts and plant extracts.

[0024] However, when the extracts are applied, the antiviral effects are mostly small, so a solution is needed. Summary of the Invention

[0025] Technical Problem

[0026] Therefore, an object of the present invention is to provide a composition having very excellent and broad antiviral activity to solve the above-mentioned problems.

[0027] Technical Solution

[0028] To solve the above-mentioned problems, the present invention provides a composition for preventing, alleviating or treating viral infections, which contains ginseng extracts and steroid compounds as active ingredients, and the ginseng extracts are selected from the group consisting of ginseng extract, red ginseng extract and black ginseng extract.

[0029] The composition of the present invention as described above is characterized in that the ginseng extracts are extracted by using water or ethanol as an extraction solvent.

[0030] The composition of the present invention as described above is characterized in that after extracting the ginseng extracts, one or more enzymes selected from the group consisting of β-galactosidase, β-glucosidase, β-glucanase, α-amylase and cellulase are used for treatment.

[0031] The composition of the present invention as described above is characterized in that the ginseng extracts are fermented using lactic acid bacteria or yeast.

[0032] The composition of the present invention as described above is characterized in that after the ginseng extracts are treated with enzymes, they are fermented using lactic acid bacteria or yeast.

[0033] The composition of the present invention as described above is characterized in that the steroid compounds are one or more selected from the group consisting of dexamethasone, hydrocortisone, prednisolone, methylprednisolone and triamcinolone.

[0034] The composition of the present invention as described above is characterized in that the virus can be any one of coronavirus, influenza virus and adenovirus.

[0035] Effects of the Invention

[0036] The composition provided by the present invention has very significant inhibitory activity against various viruses such as coronavirus, influenza virus, and adenovirus. Therefore, it can effectively prevent, improve, or treat infections caused by viruses or diseases caused by infections.

[0037] In particular, from the perspective of inhibiting viruses, the use of steroid compounds may be disadvantageous. However, even if the composition of the present invention contains steroid compounds, its antiviral effects such as virus proliferation inhibition will be significantly enhanced instead, thus solving the problems caused by the use of steroid compounds. At the same time, it has the characteristics that the useful aspects of steroid compounds for virus-infected patients can be fully utilized, such as the effect of alleviating inflammation such as pneumonia or bronchitis.

[0038] Therefore, the composition of the present invention is an excellent composition in terms of the anti-inflammatory effect that can alleviate the symptoms caused by virus infection and the antiviral effect that can inhibit virus proliferation. Detailed Embodiments

[0039] The following will describe in detail the specific content for implementing the present invention.

[0040] The present invention relates to an enzyme-treated product of ginseng extracts, a fermentation product of ginseng extracts, and steroid compounds selected from the group consisting of ginseng extracts, red ginseng extracts, and black ginseng extracts. When used together, they exhibit broad and excellent antiviral activity, and steroid compounds have excellent anti-inflammatory effects on inflammations such as pneumonia and bronchitis. Therefore, it is confirmed to be effective as a composition for preventing, improving, or treating virus infections, thus completing the present invention. Therefore, the present invention provides a composition for preventing, alleviating, or treating virus infections, which contains ginseng extracts, etc. and steroid compounds as active ingredients.

[0041] In the present invention as described above, the steroid compound refers to a lipophilic compound having a steric nucleus, such as sterols, bile acids, and sex hormones, which does not contain fatty acids and is a lipid and organic compound having a common basic structure formed by the combination of four rings (three rings composed of six carbon atoms and one ring composed of five carbon atoms), and there is no particular limitation as long as it is widely known in the technical field to which the present invention belongs. However, it is preferably one or more selected from the group consisting of dexamethasone, hydrocortisone, prednisolone, methylprednisolone, and triamcinolone.

[0042] Such steroids have strong anti-inflammatory effects and play an important role in the treatment of various diseases, including asthma exacerbation and lung diseases. They can relieve symptoms such as bronchitis and pneumonia caused by viral infections, or prevent the deterioration of the condition and are effective in treatment, etc. However, there may be a problem of delaying virus clearance due to weakened immunity. In the present invention, it is aimed to maintain the anti-inflammatory effect of steroids by using steroids together with ginseng extracts, and at the same time further enhance the antiviral effect of inhibiting virus proliferation.

[0043] In the present invention as described above, the steroid compounds have currently been approved as drugs and can be used in standard amounts. Preferably, for a 60 kg dosing subject per day, the dosage of dexamethasone is 1.5 - 10 mg, hydrocortisone is 10 - 120 mg, prednisolone or methylprednisolone is 10 - 60 mg, and triamcinolone is 8 - 16 mg.

[0044] The ginseng extracts used in the present invention generally refer to ginseng extracts (including dried ginseng or fresh ginseng extracts), red ginseng extracts, and black ginseng extracts. The preparation methods of red ginseng and black ginseng can be used without particular limitation as long as they are well-known in the technical field to which the present invention pertains. In other words, as long as they are commonly used and circulated ginseng, red ginseng, and black ginseng, they can be used without particular limitation.

[0045] Moreover, the extraction method of the present invention can be used without particular limitation as long as it is a method widely known in the technical field to which the present invention pertains. For example, methods selected from heating extraction method, cold soaking extraction method, reflux cooling extraction method, steam distillation method, ultrasonic extraction method, elution method, compression method, etc. can be used. Additionally, according to the purpose, the extract can be subjected to a conventional fractionation process and purified according to a conventional purification method.

[0046] In the present invention, preferably, an extract or a mixture thereof extracted using water or ethanol as an extraction solvent by a conventional extraction method is used as the ginseng extract.

[0047] Furthermore, the extracted primary extract can be powdered by additional processes such as vacuum distillation and freeze-drying or spray-drying, and purified fractions can be obtained by various chromatographic methods, such as silica gel column chromatography, high performance liquid chromatography, and thin layer chromatography, etc.

[0048] Thus, in the present invention, the extract can be understood to include the concepts of all extraction liquids, separated compounds, fractions, and purified products obtained at each stage of extraction, fractionation, or purification, as well as their diluted, concentrated, and dried products.

[0049] Moreover, in the present invention, ginseng extracts can be used by enzyme treatment or fermentation, or by fermentation after enzyme treatment.

[0050] The enzyme used in the present invention is preferably one or more selected from the group consisting of β-galactosidase, β-glucosidase, β-glucanase, α-amylase, and cellulase.

[0051] The strain for fermentation in the present invention can be any strain known to ferment ginseng extracts, and is preferably lactic acid bacteria or yeast.

[0052] Specifically, the ginseng extract used in the present invention can be prepared by the following steps:

[0053] Extraction step: Extracting one or more ginsengs selected from the group consisting of ginseng (dried ginseng or fresh ginseng), red ginseng, and black ginseng with hot water at 70 - 100°C, or extracting ginseng with 70% by weight of ethanol;

[0054] Enzyme treatment step: Adding an enzyme to the extract in the extraction step, and performing enzyme treatment at 25 - 65°C for 2 - 7 hours; and

[0055] Fermentation step: Adding lactic acid bacteria or yeast, preferably Saccharomyces cerevisiae, to the extract after the enzyme treatment is completed, and fermenting at 20 - 40°C for 10 - 48 hours.

[0056] The dosage of the ginseng extract as described above is not particularly limited, but based on the solid content per day for a 60 kg subject, the dosage is 2 - 5 g.

[0057] The composition of the present invention composed of the black ginseng extract and steroid compounds as described above can be administered once a day at the daily dosage as described above, but is preferably administered in two to three separate doses.

[0058] The composition of the present invention as described above is considered to exhibit antiviral activity without particular limitation, because it increases the mRNA expression level of type I interferon Ifnb1 (interferon beta) that exhibits broad antiviral activity, as well as the mRNA expression levels of genes Mx1 and Mx2 that express Mx1 (MX Dynamin Like GTPase 1) and Mx2 (MX Dynamin Like GTPase 2). For example, it exhibits antiviral activity against Flaviviridae, Adenovirinae, Coronavirinae, Reoviridae, Picornaviridae, Caliciviridae, Togaviridae, Arenaviridae, Orthomyxoviridae, Paramyxoviridae, Bunyaviridae, Rhabdoviridae, Filoviridae, Astroviridae, Bornaviridae, and Arteriviridae. Preferably, it exhibits antiviral activity against coronavirus, influenza virus, and adenovirus.

[0059] In particular, the coronavirus exhibits very excellent antiviral activity against HCoV-229E, HCoV-NL63, SARS-CoV, MERS-CoV, HCoV-OC43, HCoV-HKU1, and SARS-CoV-2.

[0060] The composition of the present invention as described above can be used in pharmaceutical compositions, food compositions, food additive compositions, feed compositions, or feed additive compositions, and the use of additives, excipients, other antiviral agents, etc. for preparing these compositions is not particularly limited in conventional procedures, and the dosage form of the composition is also not particularly limited.

[0061] Hereinafter, the present invention will be described in more detail through examples and experimental examples. These examples are only used to illustrate the present invention more specifically, and it is obvious to those skilled in the art that the scope of the present invention is not limited to these examples according to the gist of the present invention.

[0062] Examples of Ginseng Usage

[0063] Preparation of the Composition

[0064] The ginseng extract is a hot water extract, an enzymatically treated product obtained by subjecting the extract to enzymatic treatment with α - amylase after extraction, and a fermented product obtained by fermenting the extract with Saccharomyces cerevisiae after extraction.

[0065] Specifically, it is prepared by the following steps:

[0066] Extraction step: Fresh ginseng is extracted with hot water at 70 - 100°C; and

[0067] Enzymatic treatment step: An enzyme is added to the extract in the extraction step, and enzymatic treatment is carried out at 45°C for 3 hours; or

[0068] Fermentation step: Saccharomyces cerevisiae is added to the extract in the extraction step, and fermentation is carried out at 30°C for 24 hours.

[0069] The composition of the present invention is prepared by combining each ginseng extract with any one selected from the group consisting of dexamethasone, hydrocortisone, prednisolone, and triamcinolone. A composition using the ginseng extract and the steroid compound alone is used as a comparative example, and its composition is shown in Table 1 below.

[0070] Table 1

[0071]

[0072]

[0073] Test Example 1:

[0074] Preparation of Mice

[0075] In the following experiment, the inventors of the present invention purchased 8 - week - old male C57BL / 6 mice from an animal experiment center and used them for the experiment. In a laboratory environment, that is, at a temperature of 21 ± 2°C and a humidity of 50 ± 10%, they were acclimatized for 1 week under the condition of 12 hours / 12 hours (dark / light cycle) by supplying sufficient feed and water.

[0076] Next, 8-week-old C57BL / 6 mice were orally administered a combination of ginseng extract with a solid content of 22.5 mg / kg, dexamethasone 0.03 mg / kg, hydrocortisone 2.5 mg / kg, prednisolone 0.1 mg / kg, and triamcinolone 0.1 mg / kg twice a day for 2 weeks, and then sacrificed. The livers were removed for the following experiments.

[0077] Confirmation of Antiviral Effect

[0078] To confirm the antiviral effects of the compositions of the present invention prepared in the examples and the compositions of the comparative examples, the livers taken from the composition-administered group and the non-administered control group of mice were crushed using a TissueLyser II (Qiagen), and then total RNA was isolated using an RNeasy mini kit (Qiagen). Then, after reverse transcription (RT) using 2 μg of RNA with a RevertAid RT kit (EP0441, Thermo Fisher Scientific), real-time qPCR (qRT-PCR) was performed using a SensiFAST SYBR Hi-ROX kit (BIO92020, Bioline, London, UK).

[0079] The qPCR results are the results of quantifying target genes using the house-keeping gene Gapdh, which include the mRNA expression levels of type I interferon Ifnb1 (interferon β), and the mRNA expression levels of genes Mx1 and Mx2 that express proteins Mx1 (MX Dynamin Like GTPase 1) and Mx2 (MX Dynamin Like GTPase 2) showing broad antiviral activity. As shown in Table 2, the values in Table 2 refer to relative expression levels, and the expression level of the control group not administered any substance was regarded as 1.

[0080] Table 2

[0081]

[0082]

[0083] As can be confirmed from Table 2 above, in the composition of the present invention and the composition of the comparative example using ginseng extract alone, the mRNA expression levels of type I interferon Ifnb1 (interferon-β) and the genes Mx1 and Mx2 of the proteins Mx1 (MX Dynamin Like GTP ase 1) and Mx2 (MX Dynamin Like GTPase 2) that exhibit broad antiviral activity are both increased, but the degree of increase is more significant in the composition of the present invention than in the composition of the comparative example.

[0084] Moreover, in Comparative Examples 4 to 7 using steroid compounds, it is decreased instead. Therefore, from the perspective of inhibiting viruses, using steroid compounds may be disadvantageous. However, even if the composition of the present invention contains steroid compounds, the antiviral effect is significantly enhanced, thus solving the problems caused by using steroid compounds. At the same time, it has the effect of alleviating inflammation such as pneumonia or bronchitis. Therefore, it has the characteristic of being able to make full use of the useful aspects for virus-infected patients.

[0085] Furthermore, comparing the examples of the present invention, in the examples where the ginseng extract is subjected to enzymatic treatment or fermentation treatment, the degree of increase in the mRNA expression levels of type I interferon Ifnb1 (interferon-β) and the genes Mx1 and Mx2 of the proteins Mx1 (MX Dynamin Like GTPas e 1) and Mx2 (MX Dynamin Like GTPase 2) that exhibit broad antiviral activity is improved.

[0086] As described above, type I interferons (IFNs) are proteins mainly secreted by virus-infected cells. The most representative type I interferons in humans are IFN-α and IFN-β, and they exhibit broad antiviral effects (Nat Rev Immunol. 2015 Feb; 15(2): 87-103.).

[0087] Moreover, both Mx1 protein (Interferon-induced GTP-binding protein Mx1) and Mx2 protein (Interferon-induced GTP-binding protein MX2) are induced by type I interferons. The Mx proteins induced by interferons correspond to markers that are known to accumulate in the human cytoplasm and bind to invading viruses to induce virus death (Trends Microbiol. 2015 Mar; 23(3): 154-63., Microbiol Mol Biol Rev. 2013 Dec; 77(4): 551-66.).

[0088] Therefore, the above results indicate that when the composition of the present invention is administered during a viral infection, it exhibits an antiviral effect and can be used for effectively preventing, improving, or treating viral infections or diseases caused by infections.

[0089] Test Example 2:

[0090] Confirmation of Antiviral Effect through in vitro Experiment

[0091] Vero-E6 cells were cultured in DMEM(- / -). During the virus infection experiment, they were inoculated into a 12-well plate at a concentration of 5×10 5 cells / well. They were cultured in an incubator at 37°C with CO 2 for 24 hours. The cell culture medium was removed and then the cells were washed twice with 1×PBS.

[0092] After pre-treating the composition of Test Example 1 with a sample at a concentration of 100 μg / ml, it was cultured in an incubator at 37°C with CO 2 for 2 hours. Then the cell culture medium was removed for virus infection. Subsequently, the virus in Table 3 was treated at a concentration of 50 pfu / well for 1 hour. Then, after removing the cell culture medium, 1.5 ml of medium was added to each well and cultured for 72 hours. The results were confirmed. As shown in Table 3, the values in Table 3 are relative virus titers, and the virus titer of the control group without any treatment was set to 100.

[0093] Table 3

[0094]

[0095]

[0096] From the experimental results in Table 3, it can be seen that in the compositions of the examples and the comparative examples using ginseng extract alone, the viral RNA of the virus decreased in both cases. However, compared with the compositions of the comparative examples, the decrease in the composition of the present invention was more than 70%, indicating significantly higher antiviral activity.

[0097] Moreover, Comparative Examples 4 to 7 using steroid compounds showed an increase in the viral RNA of the virus, which can be said to have an adverse effect on inhibiting virus proliferation. However, when used together with ginseng extract as in the present invention, the problems occurring in Comparative Examples 4 to 7 were solved by increasing the antiviral activity of ginseng extract. Therefore, the composition of the present invention is a very useful composition with strong antiviral activity and can make full use of the anti-inflammatory effect of steroid compounds.

[0098] Moreover, when comparing the embodiments of the present invention, in the embodiments where the ginseng extract is subjected to enzymatic treatment or fermentation treatment, the degree of reduction of viral RNA is increased.

[0099] Test Example 3:

[0100] Confirmation of Antiviral Effect through in vivo Experiment

[0101] To confirm the antiviral effect in vivo, 9-week-old K18-ACE2 TG mice were used in the experiment. In a laboratory environment, that is, at a temperature of 21 ± 2 °C and a humidity of 50 ± 10%, they were acclimatized for 1 week by supplying sufficient feed and water under the condition of 12 hours / 12 hours (dark / light cycle), and were orally administered once starting from the day before virus infection in the same manner as in Experimental Example 1.

[0102] Three days after virus infection, 4 orally administered mice were sacrificed, and lung tissues were obtained from each. The virus titer in the lung tissues was measured by plaque assay. As shown in Table 4, the values in Table 4 are relative virus titers, and the virus titer of the control group without any treatment was set to 100.

[0103] Table 4

[0104]

[0105]

[0106] From the test results in Table 4, it can be seen that different from the comparative example using steroid compounds, in the composition of the embodiment of the present invention and the composition of the comparative example using ginseng extract, the virus titer is reduced. Compared with the composition of the comparative example using ginseng extract, the reduction in the composition of the present invention is more than 70%, and its antiviral activity is significantly higher.

[0107] Moreover, when comparing the embodiments of the present invention, in the embodiments where the ginseng extract is subjected to enzymatic treatment or fermentation treatment, the degree of reduction of the virus titer is increased.

[0108] Examples of Red Ginseng Usage

[0109] Preparation of the Composition

[0110] Except for using red ginseng extract, it is the same as the ginseng usage embodiment, and the comparative example of using steroid compounds alone is omitted because its test results are the same as those in the cited usage embodiment. The compositions of the examples and comparative examples are shown in Table 5 below.

[0111] Table 5

[0112] Composition Example 1 Hot Water Extract of Red Ginseng + Dexamethasone Example 2 Hot Water Extract of Red Ginseng + Hydrocortisone Example 3 Hot Water Extract of Red Ginseng + Prednisolone Example 4 Hot Water Extract of Red Ginseng + Triamcinolone Example 5 Enzyme-Treated Product of Hot Water Extract of Red Ginseng + Dexamethasone Example 6 Enzyme-Treated Product of Hot Water Extract of Red Ginseng + Hydrocortisone Example 7 Enzyme-Treated Product of Hot Water Extract of Red Ginseng + Prednisolone Example 8 Enzyme-Treated Product of Hot Water Extract of Red Ginseng + Triamcinolone Example 9 Fermentation Product of Hot Water Extract of Red Ginseng + Dexamethasone Example 10 Fermentation Product of Hot Water Extract of Red Ginseng + Hydrocortisone Example 11 Fermentation Product of Hot Water Extract of Red Ginseng + Prednisolone Example 12 Fermentation Product of Hot Water Extract of Red Ginseng + Triamcinolone Comparative Example 1 Hot Water Extract of Red Ginseng Comparative Example 2 Enzyme-Treated Product of Hot Water Extract of Red Ginseng Comparative Example 3 Fermentation Product of Hot Water Extract of Red Ginseng

[0113] Test Example 1:

[0114] Preparation of Mice

[0115] In the following experiment, the inventors of the present invention purchased 8-week-old male C57BL / 6 mice from an animal experiment center and used them for the experiment. In a laboratory environment, i.e., at a temperature of 21 ± 2 °C and a humidity of 50 ± 10%, they were acclimated for 1 week by supplying sufficient feed and water under the condition of 12 hours / 12 hours (dark / light cycle).

[0116] Next, a combination of red ginseng extract with a solid content of 22.5 mg / kg, dexamethasone 0.03 mg / kg, hydrocortisone 2.5 mg / kg, prednisolone 0.1 mg / kg, and triamcinolone 0.1 mg / kg was orally administered to 8-week-old C57BL / 6 mice twice a day for 2 weeks, and then they were sacrificed and the livers were removed for the following experiments.

[0117] Confirmation of Antiviral Effect

[0118] To confirm the antiviral effects of the compositions of the present invention prepared in the examples and the compositions of the comparative examples, the livers taken from the composition-administered group and the non-administered control group of mice were ground with a TissueLyser II (Qiagen), and then total RNA was isolated using an RNeasy mini kit (Qiagen). Then, after reverse transcription (RT) using 2 μg of RNA with a RevertAid RT kit (EP0441, Thermo Fisher Scientific), real-time qPCR (qRT-PCR) was performed using a SensiFAST SYBR Hi-ROX kit (BIO92020, Bioline, London, UK).

[0119] The qPCR results are the results of quantifying target genes by the house-keeping gene Gapdh, which include the mRNA expression level of type I interferon Ifnb1 (interferon-β), and the mRNA expression levels of genes Mx1 and Mx2 that express proteins Mx1 (MX Dynamin Like GTPase 1) and Mx2 (MX Dynamin Like GTPase 2) showing broad antiviral activities. As shown in Table 6, the values in Table 6 refer to relative expression levels, and the expression level of the control group without administration of any substance is regarded as 1.

[0120] Table 6

[0121]

[0122]

[0123] It can be confirmed from Table 6 above that in the compositions of the present invention and the comparative composition using red ginseng extract alone, the mRNA expression level of type I interferon Ifnb1 (interferon-β) and the mRNA expression levels of genes Mx1 and Mx2 that express proteins Mx1 (MX Dynamin Like GTPase 1) and Mx2 (MX Dynamin Like GTPase 2) showing broad antiviral activities all increased, but the degree of increase was more significant in the composition of the present invention than in the comparative composition. Comparing the examples of the present invention, in the examples where red ginseng extract was subjected to enzymatic treatment or fermentation treatment, the degree of increase was further improved.

[0124] Moreover, compared with the example of using ginseng, in the example of using red ginseng as this example, the mRNA expression level of type I interferon Ifnb1 (interferon-β) and the mRNA expression levels of genes Mx1 and Mx2 that express proteins Mx1 (MX DynaminLike GTPase 1) and Mx2 (MX Dynamin Like GTPase 2) showing broad antiviral activities generally increased, which means that red ginseng is superior to ginseng in terms of antiviral activity.

[0125] As described above, type I interferons (IFNs) are proteins mainly secreted by virus-infected cells. The most representative type I interferons in humans are IFN-α and IFN-β, and they exhibit broad antiviral effects (Nat Rev Immunol. 2015 Feb; 15(2): 87-103.).

[0126] Furthermore, both Mx1 protein (Interferon-induced GTP-binding protein Mx1) and Mx2 protein (Interferon-induced GTP-binding protein MX2) are induced by type I interferons. The interferon-induced Mx proteins correspond to markers known to accumulate in the human cytoplasm and bind to invading viruses to induce viral death (Trends Microbiol. 201

[0127] 5 Mar; 23(3):154-63., Microbiol Mol Biol Rev. 2013 Dec; 77(4):551-66.).

[0128] Therefore, the above results indicate that when the composition of the present invention is administered during a viral infection, it exhibits an antiviral effect and can be used to effectively prevent, ameliorate, or treat viral infections or diseases caused by infection.

[0129] Test Example 2:

[0130] Confirmation of Antiviral Effect through in vitro Experiment

[0131] Vero-E6 cells were cultured in DMEM(- / -). During the viral infection experiment, they were inoculated into 12-well plates at a concentration of 5×10 5 cells / well. They were cultured in an incubator at 37°C with CO 2 for 24 hours. The cell culture medium was removed and then the cells were washed twice with 1×PBS.

[0132] After pre-treating the composition of Test Example 1 with a sample at a concentration of 100 μg / ml, it was cultured in an incubator at 37°C with CO 2 for 2 hours. Then the cell culture medium was removed for viral infection. Subsequently, the viruses in Table 7 were treated at a concentration of 50 pfu / well for 1 hour. Then, after removing the cell culture medium, 1.5 ml of medium was added to each well and cultured for 72 hours. The results were then confirmed. As shown in Table 7, the values in Table 7 are relative virus titers, with the virus titer of the control group without any treatment set to 100.

[0133] Table 7

[0134]

[0135]

[0136] From the experimental results in Table 7, it can be seen that in the compositions of the examples and the comparative examples using red ginseng extract alone, the viral RNA of the virus decreased. However, compared with the compositions of the comparative examples, the decrease in the compositions of the present invention was more than 70%, indicating significantly higher antiviral activity. Comparing the examples of the present invention, in the examples where the red ginseng extract was subjected to enzymatic treatment or fermentation treatment, the degree of decrease in viral RNA was increased.

[0137] Moreover, compared with the examples using ginseng, in the examples using red ginseng as in this example, the degree of decrease in viral RNA was generally increased, which means that red ginseng is superior to ginseng in terms of antiviral activity.

[0138] Test Example 3:

[0139] Confirmation of Antiviral Effect through in vivo Experiment

[0140] To confirm the in vivo antiviral effect, 9-week-old K18-ACE2 TG mice were used in the experiment. In the laboratory environment, that is, at a temperature of 21 ± 2 °C and a humidity of 50 ± 10%, they were acclimated for 1 week by supplying sufficient feed and water under the condition of 12 hours / 12 hours (dark / light cycle), and were orally administered once starting from the day before virus infection in the same manner as in Test Example 1.

[0141] Three days after virus infection, 4 orally administered mice were sacrificed, and lung tissues were obtained from each. The virus titer in the lung tissues was measured by plaque assay. As shown in Table 8, the values in Table 8 are relative virus titers, and the virus titer of the control group without any treatment was set to 100.

[0142] Table 8

[0143]

[0144]

[0145] From the test results in Table 8, it can be seen that in the compositions of the examples of the present invention and the compositions of the comparative examples using red ginseng extract, the virus titers decreased. Compared with the compositions of the comparative examples using red ginseng extract, the degree of decrease in the compositions of the present invention was significantly increased. Comparing the examples of the present invention, in the examples where the red ginseng extract was subjected to enzymatic treatment or fermentation treatment, the degree of decrease in virus titer was increased.

[0146] Moreover, compared with the examples using ginseng, in the examples using red ginseng as in this example, the degree of decrease in virus titer was generally increased, which means that red ginseng is superior to ginseng in terms of antiviral activity.

[0147] Examples of Black Ginseng Usage

[0148] Preparation of the Composition

[0149] Except for using black ginseng extract, it is the same as the ginseng usage example. Also, the comparative example of using only steroid compounds is omitted because its test results are the same as those in the cited usage examples. The compositions of the examples and comparative examples are shown in Table 9 below.

[0150] Table 9

[0151]

[0152]

[0153] Test Example 1:

[0154] Preparation of Mice

[0155] In the following experiment, the inventors of the present invention purchased 8-week-old male C57BL / 6 mice from an animal experiment center and used them for the experiment. In a laboratory environment, that is, at a temperature of 21 ± 2 °C and a humidity of 50 ± 10%, they were acclimated for 1 week by supplying sufficient feed and water under the condition of 12 hours / 12 hours (dark / light cycle).

[0156] Next, an 8-week-old C57BL / 6 mouse was orally administered a combination of black ginseng extract with a solid content of 22.5 mg / kg, dexamethasone 0.03 mg / kg, hydrocortisone 2.5 mg / kg, prednisolone 0.1 mg / kg, and triamcinolone 0.1 mg / kg twice a day for 2 weeks, and then sacrificed. The liver was removed for the following experiments.

[0157] Confirmation of Antiviral Effect

[0158] To confirm the antiviral effects of the compositions of the present invention prepared in the examples and the compositions of the comparative examples, the livers taken from the composition-administered group and the non-administered control group of mice were pulverized using a TissueLyser II (Qiagen), and then total RNA was isolated using an RNeasy mini kit (Qiagen). Then, after reverse transcription (RT) using 2 μg of RNA with a RevertAid RT kit (EP0441, Thermo Fisher Scientific), real-time qPCR (qRT-PCR) was performed using a SensiFAST SYBR Hi-ROX kit (BIO92020, Bioline, London, UK).

[0159] The qPCR results are the results of quantifying target genes using the house-keeping gene Gapdh, and include the mRNA expression levels of type I interferon Ifnb1 (interferon-β), and the mRNA expression levels of genes Mx1 and Mx2 that express proteins Mx1 (MX Dynamin Like GTPase 1) and Mx2 (MX Dynamin Like GTPase 2) which exhibit broad antiviral activities. As shown in Table 10, the values in Table 10 refer to relative expression levels, and the expression level of the control group that was not administered any substance was regarded as 1.

[0160] Table 10

[0161]

[0162] It can be confirmed from Table 10 above that in the compositions of the present invention and the compositions of the comparative examples using only black ginseng extract, the mRNA expression levels of type I interferon Ifnb1 (interferon-β), and the mRNA expression levels of genes Mx1 and Mx2 that express proteins Mx1 (MX Dynamin Like GTPase 1) and Mx2 (MX Dynamin Like GTPase 2) which exhibit broad antiviral activities all increased, but the degree of increase was more significant in the compositions of the present invention than in the compositions of the comparative examples. Comparing the examples of the present invention, in the examples where the black ginseng extract was subjected to enzymatic treatment or fermentation treatment, the degree of increase was further enhanced.

[0163] Moreover, compared with the ginseng usage example and the red ginseng usage example, in the black ginseng usage example of this embodiment, the mRNA expression level of type I interferon Ifnb1 (interferon-β) and the genes Mx1 and Mx2 of the proteins Mx1 (MX Dynamin Like GTPase 1) and Mx2 (MX Dynamin Like GTPase 2) that exhibit broad antiviral activity showed an overall increase in mRNA expression level, indicating that black ginseng is superior to ginseng and red ginseng in terms of antiviral activity.

[0164] As described above, type I interferons (IFNs) are proteins mainly secreted by virus-infected cells. The most representative type I interferons in humans are IFN-α and IFN-β, and they exhibit broad antiviral effects (Nat Rev Immunol. 2015 Feb; 15(2):87-103.).

[0165] Moreover, both Mx1 protein (Interferon-induced GTP-binding protein Mx1) and Mx2 protein (Interferon-induced GTP-binding protein MX2) are induced by type I interferon. The interferon-induced Mx proteins correspond to markers that are known to accumulate in the human cytoplasm and bind to invading viruses to induce virus death (Trends Microbiol. 2015 Mar; 23(3):154-63., Microbiol Mol Biol Rev. 2013 Dec; 77(4):551-66.).

[0166] Therefore, the above results indicate that when the composition of the present invention is administered during virus infection, it exhibits antiviral effects and can be used for effectively preventing, improving, or treating virus infections or diseases caused by infections.

[0167] Test Example 2:

[0168] Confirmation of Antiviral Effect through in vitro Experiment

[0169] Vero-E6 cells were cultured in DMEM(- / -). During the virus infection experiment, they were inoculated into 12-well plates at a concentration of 5×10 5 cells / well. They were cultured in an incubator at 37°C and CO 2 for 24 hours. The cell culture medium was removed, and then they were washed twice with 1×PBS.

[0170] After pre-treating the composition of Test Example 1 with a sample at a concentration of 100 μg / ml, at 37°C and CO2 Incubate in an incubator for 2 hours, then remove the cell culture medium for virus infection, and then treat with the virus in Table 11 at a concentration of 50 pfu / well for 1 hour. Then, after removing the cell culture medium, add 1.5 ml of medium to each well and incubate for 72 hours. Then confirm the results. As shown in Table 11, the values in Table 11 are relative virus titers, and the virus titer of the control group without any treatment is set to 100.

[0171] Table 11

[0172]

[0173]

[0174] From the test results in Table 11, it can be seen that in the compositions of the examples and the comparative examples using black ginseng extract alone, the viral RNA of the virus decreased, but compared with the compositions of the comparative examples, the antiviral activity of the composition of the present invention was significantly higher. Comparing the examples of the present invention, in the examples where the black ginseng extract was enzymatically treated or fermented, the degree of reduction of viral RNA was improved.

[0175] Moreover, compared with the examples of using ginseng and red ginseng, in the example of using black ginseng as the present example, the degree of reduction of viral RNA was generally improved, which means that black ginseng is superior to ginseng and red ginseng in terms of antiviral activity.

[0176] Test Example 3:

[0177] Confirmation of Antiviral Effect through in vivo Experiment

[0178] To confirm the in vivo antiviral effect, 9-week-old K18-ACE2 TG mice were used in the experiment. In the laboratory environment, that is, at a temperature of 21 ± 2 °C and a humidity of 50 ± 10%, acclimatize for 1 week by supplying sufficient feed and water under the condition of 12 hours / 12 hours (dark / light cycle), and orally administer once starting from the day before virus infection in the same manner as in Experimental Example 1.

[0179] Three days after virus infection, 4 orally administered mice were sacrificed, and lung tissues were obtained from each. The virus titer in the lung tissues was measured by plaque assay. As shown in Table 12, the values in Table 12 are relative virus titers, and the virus titer of the control group without any treatment is set to 100.

[0180] Table 12

[0181]

[0182] From the experimental results in Table 12, it can be seen that in the composition of the embodiment of the present invention and the composition of the comparative example using the black ginseng extract, the virus titer decreased. Compared with the composition of the comparative example using the black ginseng extract, the degree of decrease in the composition of the present invention was significantly improved. Comparing the embodiments of the present invention, in the embodiments where the black ginseng extract was subjected to enzymatic treatment or fermentation treatment, the degree of decrease in the virus titer was increased.

[0183] Moreover, compared with the embodiment of using ginseng and the embodiment of using red ginseng, in the embodiment of using black ginseng as the present embodiment, the degree of decrease in the virus titer was generally increased, which means that black ginseng is superior to ginseng and red ginseng in terms of antiviral activity.

[0184] In summary, the composition of the present invention can be used as a therapeutic agent for various virus-induced infections or infectious diseases.

[0185] Therefore, the composition provided by the present invention has very significant inhibitory activity against various viruses such as Corona virus, Influenzavirus, and Adeno virus, and thus can effectively prevent, improve, or treat virus-induced infections or diseases caused by infections.

[0186] Furthermore, the present invention has the advantage of eliminating side effects such as weakened immunity that may be caused by steroid compounds by using steroid compounds and ginseng extracts together.

[0187] Therefore, the composition of the present invention is an excellent composition in terms of both the anti-inflammatory effect that can relieve symptoms caused by virus infection and the antiviral effect that can inhibit virus proliferation.

Claims

1. A pharmaceutical composition for preventing, alleviating or treating viral infections, characterized in that, it contains ginseng extracts and steroid compounds as active ingredients, and the ginseng extracts are selected from the group consisting of ginseng extract, red ginseng extract and black ginseng extract.

2. A health food composition for preventing or improving viral infections, characterized in that, it contains ginseng extracts and steroid compounds as active ingredients, and the ginseng extracts are selected from the group consisting of ginseng extract, red ginseng extract and black ginseng extract.

3. A feed composition for preventing or improving viral infections, characterized in that, it contains ginseng extracts and steroid compounds as active ingredients, and the ginseng extracts are selected from the group consisting of ginseng extract, red ginseng extract and black ginseng extract.

4. The composition according to any one of claims 1, 2 and 3, characterized in that, the virus is any one of coronavirus, influenza virus and adenovirus.

5. The composition according to any one of claims 1, 2 and 3, characterized in that, the ginseng extracts are extracted by using water or ethanol as an extraction solvent.

6. The composition according to claim 5, characterized in that, after extracting the ginseng extracts, one or more enzymes selected from the group consisting of β-galactosidase, β-glucosidase, β-glucanase, α-amylase and cellulase are used for treatment.

7. The composition according to claim 5, characterized in that, after extracting the ginseng extracts, lactic acid bacteria or yeast are used for fermentation.

8. The composition according to claim 6, characterized in that, after the ginseng extracts are treated with enzymes, lactic acid bacteria or yeast are used for fermentation.

9. The composition according to any one of claims 1, 2 and 3, characterized in that, the steroid compounds are one or more selected from the group consisting of dexamethasone, hydrocortisone, prednisolone, methylprednisolone and triamcinolone.

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