KR20220035675A - Phamaceutical composition for antiviral and health functional food composition using gynostemma pentaphyllum extract - Google Patents

Abstract

The present invention relates to an antiviral pharmaceutical composition and a health functional food composition containing a Gynecnospermia extract as an active ingredient, and which contains a Gynostiasis extract prepared by solvent extraction of Gynecnospermia powder as an active ingredient.
As described above, antiviral pharmaceutical compositions and health functional food compositions containing Gynostrum extract as an active ingredient not only exhibit excellent antiviral effects, but also have low cytotoxicity, so they can be safely used even when taken for a long period of time for preventive purposes.

Description

Antiviral pharmaceutical composition and health functional food composition containing Gynostemma extract as an active ingredient {PHAMACEUTICAL COMPOSITION FOR ANTIVIRAL AND HEALTH FUNCTIONAL FOOD COMPOSITION USING GYNOSTEMMA PENTAPHYLLUM EXTRACT}

The present invention relates to an antiviral pharmaceutical composition and a health functional food composition containing a Gynectarsus extract as an active ingredient, and more specifically, to an antiviral pharmaceutical composition and a health functional food composition that not only exhibits excellent antiviral effects but also has low cytotoxicity, making it suitable for long-term use for preventive purposes. It relates to an antiviral pharmaceutical composition and health functional food composition containing Gynostrum extract as an active ingredient that can be safely used.

Influenza is emerging as a serious medical, economic, and social problem for humanity, and it is estimated that more than 500 million people around the world contract the flu every year, and about 2 million of them die. In particular, the 'Spanish flu' that occurred during World War I in 1918 caused such a huge loss of life that it could be called a modern-day Black Death. It is estimated that approximately 20 to 50 million people died between 1918 and 1919.

According to the 'Status of New Infectious Disease Occurrence and Damage' report recently published by the Science and Technology Planning and Evaluation Institute, the global society has been affected by the outbreak of six major new infectious diseases (SARS, H1N1 influenza, Avian influenza, Ebola virus, MERS, and Zika virus) since 2000. And economic losses were estimated at $800 billion (approximately 864 trillion won). By new infectious disease, severe respiratory syndrome (SARS, 2003) infected 8,098 people in 29 countries, and swine flu (H1N1, 2009) infected more than 130,000 people worldwide. Avian influenza (H7N9, AI, 2013) human infections mainly occurred in China. In particular, since October 2016, 429 patients with AI human infection have appeared, and early last year, 99 people died, with a fatality rate of 40%. The fatality rate of Ebola virus (Ebola hemorrhagic fever, 2014) is 41%, which is no less than that of AI human infectious disease. It was found that 24,509 people were infected with Ebola virus, which is mainly prevalent in Africa. In addition, Middle East Respiratory Syndrome (MERS), which caused massive damage in a domestic epidemic in 2015, occurred in 12 countries, including the Middle East and North Africa, and 1,040 people were infected, with a high fatality rate of 37%.

However, despite the existence of a large number of anti-influenza drugs, the number of influenza epidemics each year still remains high, and although recently developed drugs are highly effective against currently circulating influenza virus strains, the rate of resistance of the virus to drugs or vaccines is limited. It is evolving faster than the speed of development. In this regard, there is a need for new therapeutic approaches and drugs with a broad spectrum of activity.

Accordingly, experimental and clinical research is being conducted at the in vitro and in vivo levels on various types of drugs showing effectiveness in combating influenza infection, but the reality is that drug development remains at a basic level compared to the speed of emergence of new viruses.

Recently, known antiviral drugs such as lopinavir/ritonavir, darunavir/umifenovir, oseltamivir, favipiravir, remdesivir, chloroquine, hydroxychloroquine, azithromycin, tocilizumab, and interpheron-β are being actively studied as treatments for SARS virus and COVID-19. It is becoming. Currently, no vaccine or treatment for COVID-19 has been developed, and the development of treatments using natural products or drug re-creation methods using existing drugs is actively underway.

Natural products with the most antiviral activity include polyphenols and flavonoids (quercetin, luteolin, hesperetin, amentoflavone, tetra-O-gallyl-β-D-glucose, sinigrin, forsythoside A, psoralidin, tomentin B, terrestrimine, broussochalcone, papyriflavonol A) ) is known. In addition, alkaloids (lycorine, tylophorine, 7-methoxycryptopleurine, jubanine H, nummularine B), anthraquinones (aloe-emodin, emodin), saponins (glycryrrhizin, escinidin, saikosaponin B2), and terpenes ) (curcumin, betulinic acid, savinin, iguesterin, dihydrotanshinone I, cryptotanshinone, 3β-friedelanol, chrysanthemumin B), coumarins (leptodactylone, xanthoangelol E), diarylheptanoids (hirsutenone), and lectins ( APA, UDA, HHA, alstotide 1), etc. are known.

Viruses are particles with a diameter of 80 to 100 nm coated with a lipid membrane, an integrated surface of three types of glycoproteins, hemagglutinin (HA), neuraminidase (NA), and the viral ion channel (M2), which contain genes. If you look at the structure. Ribonucleoproteins (RNPs) are composed of eight, single-stranded negative polarity RNAs in complex with three subunits of the nuclear protein and polymerase complex. After the virus penetrates the cell, ribonucleoproteins reach the nucleus, where transcription, translation, and replication of the viral genome segment occur.

Transcription and replication of virus-specific RNA is performed by the viral polymerase complex. Unlike eukaryotic polymerases, viral polymerases do not have an error correction mechanism. Therefore, the mutation frequency of the viral genome is, according to various estimates, 10 ^-4 to 10 ^-6 nucleotides per replication cycle, which is several times higher than the mutation rate of bacteria and eukaryotes, and due to the short replication period, influenza viruses, etc. are prone to mutations, etc. The same mutation occurs quickly. The rapid emergence of mutations renders the virus incapable of the host's immune response and defense network. Therefore, despite the formation of an immune layer in the population due to vaccination and natural outbreaks, epidemics can occur every year. Additionally, as a result of the use of antiviral drugs, drug-resistant strains of the virus have emerged, reducing the effectiveness of antiviral chemotherapy.

Meanwhile, Gynostemma pentaphyllum (Thunb.) Makino} is included in the genus Gynostemma Bl of the Cucurbitaceae family. Widely distributed in Northeast Asia and Southeast Asia. G. pentaphyllum has been used in food and supplement products for hundreds of years in China and is mainly distributed in the Qingling Mountains and south of the Yangtze River. According to Oriental medicine, the taste and nature of G. pentaphyllum are slightly bitter, neutral, and warm. G. pentaphyllum has been used in the private sector since the Ming Dynasty to treat hematuria, edema, pharyngeal pain, neck heat and swelling, tumors and trauma. Currently, many of Ddol's products, including tea, tablets, instant powder, capsules, oral liquid, and tablets, are being released in the United States, China, and Europe, and are used as additives in beverages, sports drinks, cola, beer, biscuits, bread, and noodles. A lot of research is being done for this purpose.

Republic of Korea Patent Registration No. 1047755 (2011.07.01) Republic of Korea Patent Registration No. 2092832 (2020.03.18)

The object of the present invention is to provide an antiviral pharmaceutical composition and health functional food composition containing Gynostrum extract as an active ingredient, which not only exhibits excellent antiviral effect, but also has low cytotoxicity and can be safely used even when taken for a long period of time for preventive purposes. It is provided.

The object of the present invention is achieved by providing an antiviral pharmaceutical composition containing a Gynecnospermia extract as an active ingredient, which is characterized in that it contains a Gynecnospermia extract as an active ingredient.

According to a preferred feature of the present invention, the antiviral pharmaceutical composition containing the Gynecnospermia extract as an active ingredient contains 5 to 40 μg/mL of the Gynecnospermia extract.

According to a more preferred feature of the present invention, the Gynostemma extract is contained in an amount of 10 to 20 μg/mL.

According to a more preferred feature of the present invention, the Gynostrum extract induces antiviral factors TNF-α, IL-1β, and IL-6.

According to a more preferred feature of the present invention, the Gynecnospermia extract is prepared by mixing 100 parts by weight of Gynecdotes powder with 500 to 1,500 parts by weight of a solvent consisting of at least one selected from the group consisting of water and alcohols having 1 to 4 carbon atoms, and then mixing 20 to 30 parts by weight at room temperature. It is manufactured by extraction over time.

According to an even more preferred feature of the present invention, the Gynecnospermia extract is prepared by mixing 1000 parts by weight of ethanol with 100 parts by weight of Gynecnospermia powder and extracting at room temperature for 24 hours.

According to an even more preferred feature of the invention, the virus is an influenza virus.

According to an even more preferred feature of the present invention, the antiviral pharmaceutical composition containing the Gynecnophyllum extract as an active ingredient further includes a pharmaceutically acceptable carrier, excipient, or diluent.

According to an even more preferred feature of the present invention, the antiviral pharmaceutical composition containing the Gynecnophyllum extract as an active ingredient is available in tablets, pills, powders, granules, capsules, suspension emulsions, syrups, aerosols, topical preparations, suppositories, and injections. It has a dosage form selected from the group consisting of:

In addition, the object of the present invention can be achieved by providing an antiviral health functional food composition containing Gynecnospermia extract as an active ingredient, which is characterized in that it contains Gynecnospermia extract as an active ingredient.

According to a preferred feature of the present invention, the health functional food has one or more dosage forms selected from the group consisting of powder, granule, tablet, capsule, candy, chewing gum, jelly and beverage.

The antiviral pharmaceutical composition and health functional food composition containing Gynostrum extract as an active ingredient according to the present invention not only exhibits excellent antiviral effects, but also has low cytotoxicity, making it an excellent product that can be safely used even when taken for a long period of time for preventive purposes. Shows effect.

Figure 1 is a graph showing the toxicity measured by applying the Gynostrum extract prepared through Preparation Example 1 of the present invention to cells cultured through Preparation Example 2 and then treating them with MTS reagent.
Figure 2 is a photograph showing cytotoxicity taken under a microscope after applying the Gynostrum extract prepared through Preparation Example 1 of the present invention to cells cultured through Preparation Example 2.
Figure 3 is a photograph showing the treatment of Gynostrum extract prepared through Preparation Example 1 of the present invention to cells cultured through Preparation Example 2, then infection with influenza virus (PR8-GFP) and observation under a fluorescence microscope (Mock; Negative) Control group, PR8-GFP; Influenza virus infection group, PR8-GFP/IFN-β; Interferon beta-treated group, PR8-GFP/Gyptoids 10 ㎍/mL; Gypsypodia 10 ㎍/mL treated group, R8-GFP/Gynostemsi 20 ㎍/mL mL; 20㎍/mL treatment group).
Figures 4 and 5 show the results of confirming the antiviral efficacy of the Gynostrum extract prepared through Preparation Example 1 of the present invention against influenza virus (PR8-GFP) using the CCK-8 assay method, and Figure 4 shows the results of the CCK-8 reagent. is a photograph of cells treated, and Figure 5 is a graphical graph quantifying Figure 4. The cell survival rate of the negative control group that was not treated with the virus was set at 100%, and the relative cell survival rate was calculated (Mock; negative control group, PR8- GFP; Influenza virus infection group, PR8-GFP/IFN-β; Interferon beta-treated group, PR8-GFP/Gyptoids 5 ㎍/mL; Gypsum 5 ㎍/mL treated group, PR8-GFP/Gynostemsi 10 ㎍/mL; Gypsum 10 ㎍/mL treatment group, R8-GFP/Gynostemma 20㎍/mL; Gynecostasis 20㎍/mL treatment group).
Figures 6 to 8 show the results of observing the antiviral efficacy under a fluorescence microscope after co-infecting the Gynostrum extract prepared through Preparation Example 1 of the present invention with influenza virus (PR8-GFP) (Mock; negative control, PR8-GFP; Influenza virus infection group, PR8-GFP/IFN-β; interferon beta-treated group, PR8-GFP/Gyptoids 10 μg/mL; Gypsum 10 μg/mL-treated group, R8-GFP/Gyptoids 20 μg/mL; Gypsum 20 μg/mL mL treatment group), Figure 6 is a photograph taken with a fluorescence microscope, Figure 7 is a histogram confirming GFP expression in each cell using a flow cytometer, and Figure 8 shows the fluorescence value of the GFP protein expressed through flow cytometry. This is a graph that is quantified and compared relative to the fluorescence value caused by the PR8-GFP virus.
Figure 9 shows RAW 264.7 cells cultured through Preparation Example 2 of the present invention were simultaneously infected with 10 or 20 ㎍/mL of the Gynostrum extract prepared through Preparation Example 1 and PR8-GFP virus, then the cells were collected, proteins were separated, and influenza This is the result of comparing the expression levels of viral proteins HA, PA, and M2 (HA; Hamaglutinin A, M2; Matrix 1 protein).
Figure 10 shows RAW 264.7 cells cultured through Preparation Example 2 of the present invention were simultaneously infected with 20 ㎍/mL of the Gynostrum extract prepared through Preparation Example 1 and PR8-GFP virus, and then the cells were fixed and infected with the viral proteins HA, M2, This is the result of confirming the expression of NA, NP, NS1, and PA using immunofluorescence staining. DAPI stains the nucleus of the cell, and Merge is the result of simultaneously marking the location of the nucleus and viral protein (-; negative control group, PR8-GFP; influenza virus infection group, PR8-GFP/Goldoe; Gyptoe 20㎍/mL treated group ), (NA; Neuraminidase, NP; Nucleoprotein, NS1 (Non-structural protein 1).
Figures 11 to 13 are graphs showing anti-viral cytokine mRNA treated with Gynostrum extract PR8-GFP virus prepared in Preparation Example 1 in RAW 264.7 cells cultured in Preparation Example 2 of the present invention analyzed by qPCR.

Below, preferred embodiments of the present invention and the physical properties of each component are described in detail, but the purpose is to provide a detailed description so that a person skilled in the art can easily carry out the invention. This does not mean that the technical idea and scope of the present invention are limited.

The antiviral pharmaceutical composition containing the Gynecnospermia extract as an active ingredient according to the present invention contains the Gynecnospermia extract as an active ingredient, and it is preferable that the Gynecnospermia extract is contained in an amount of 5 to 40 μg/mL.

If the Gynecnospermia extract is contained in less than 5 μg/mL, the antiviral effect is minimal, and if the Gynecnospermia extract is contained in excess of 40 μg/mL, it is undesirable because it exhibits cytotoxicity. It is most desirable to contain it in /mL.

As shown in Figures 11 to 13 below, the Gynostrum extract induces the antiviral factors TNF-α, IL-1β and IL-6, especially when the Gynostrum extract is contained at 10 to 20 μg/mL, the It can be seen that the induction effect of antiviral factors is significantly improved.

In addition, the ginseng extract is prepared by mixing 100 parts by weight of ginseng ginseng powder, which is prepared by drying and pulverizing ginseng ginseng, with 500 to 1,500 parts by weight of a solvent consisting of at least one selected from the group consisting of water and alcohols with a carbon number of 1 to 4, and mixing them at room temperature. It is manufactured through an extraction process for 20 to 30 hours, and is preferably manufactured by mixing 1000 parts by weight of ethanol with a mass concentration of 70% with 100 parts by weight of Gynecnospermia powder and extracting it at room temperature for 24 hours.

In addition, the Gynostrum extract prepared through the above process may be applied in the form of any one of a diluted or concentrated extract, a dried product obtained by drying the extract, a crude product, or a purified product.

In recent decades, pharmacological research has been conducted on the efficacy of Gyllium ginseng, including antibacterial, anti-cancer, anti-aging, anti-fatigue, anti-ulcer, hypolipidemia, and immunomodulatory activities. The various pharmacological effects of ginseng include saponins, amino acids, polysaccharides, It originates from a variety of chemical components, including flavonoids, organic acids, trace elements, and other chemicals.

In addition, examples of viruses in which the above-mentioned Gynostrum extract has antiviral activity include Orthomixoviridae, Rhabdoviridae, Paramixoviridae, Herpesviridae, and Picona. It is one or more viruses selected from Picornaviridae, and is preferably an influenza virus.

In addition, the antiviral pharmaceutical composition containing the Gynecnospermia extract as an active ingredient may further include a pharmaceutically acceptable carrier, excipient, or diluent. The types of the pharmaceutically acceptable carrier, excipient, or diluent are described in the present invention. As long as it does not impair the effect of the antiviral pharmaceutical composition according to the above, there is no particular limitation and any one can be used, for example, fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, sweeteners, fragrances, preservatives. It may include etc. Representative examples of pharmaceutically acceptable carriers, excipients or diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, gelatin, glycerin, gum acacia, alginate, calcium phosphate, calcium carbonate, calcium. Silicates, Cellulose, Methyl Cellulose, Microcrystalline Cellulose, Polyvinyl Pyrrolidone, Water, Methylhydroxybenzoate, Propylhydroxybenzoate, Talc, Magnesium Stearate, Mineral Oil, Propylene Glycol, Polyethylene Glycol, Vegetable Oil, Injectable Examples include ester, Witepsol, Macrogol, Tween 61, Kakaoji, and Lauridge.

In addition, the antiviral pharmaceutical composition containing the Gynecnospermia extract as an active ingredient according to the present invention is selected from the group consisting of tablets, pills, powders, granules, capsules, suspension emulsions, syrups, aerosols, external preparations, suppositories, and injections. It can be prepared in a dosage form, and the method of formulating the antiviral pharmaceutical composition according to the present invention can be carried out according to a conventional method known in the art, and is not particularly limited.

In addition, the antiviral pharmaceutical composition containing the Gynostrum extract as an active ingredient according to the present invention can be administered orally or parenterally, and the dosage is determined by the age, gender, weight, condition, degree of disease, and drug use of the administering subject. It can be appropriately selected depending on the form, route and period of administration, and the formulation method, dosage, route of administration, components, etc. can be appropriately selected from conventional techniques known in the art.

In addition, the antiviral pharmaceutical composition containing the Gynostiasis extract as an active ingredient according to the present invention can be used for the prevention and treatment of bacterial infectious diseases or viral infectious diseases, and can contain other pharmaceutically active ingredients in addition to the Gynostiasis extract as an active ingredient. It can be used together or mixed with a pharmaceutical composition containing other active ingredients.

In addition, the antiviral health functional food composition containing Gynecnospermia extract as an active ingredient according to the present invention contains Gynecnospermia extract as an active ingredient.

In addition, the antiviral health functional food composition containing Gynostrum extract as an active ingredient according to the present invention may further include a foodologically acceptable food supplement. Foodologically acceptable food supplements that can be used in the present invention include natural carbohydrates such as sugars such as glucose, fructose, maltose, sucrose, dextrin, and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol, and tau. Natural flavors such as martin and stevia extract, synthetic flavors such as saccharin and aspartamic acid, colorants, pectic acid or its salts, alginic acid or its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin , alcohol, carbonating agent, etc., but is not limited thereto.

In addition, the antiviral health functional food composition containing the Gynecnospermia extract as an active ingredient according to the present invention may be in a form selected from the group consisting of powder, granule, tablet, capsule, candy, chewing gum, jelly, and beverage. The content of Gynostrum extract in the health functional food composition for viruses may be appropriately selected considering the form, flavor, and taste of the food, and may range from 0.01 to 30% by weight based on the total weight of the health functional food, for example. .

In addition, the form, composition, manufacturing method, etc. of the antiviral health functional food composition containing the Gynostrum extract as an active ingredient according to the present invention may be appropriately selected from conventional techniques known in the technical field.

Hereinafter, the method for producing an antiviral pharmaceutical composition containing the Gynecnospermia extract as an active ingredient according to the present invention and the physical properties of the antiviral pharmaceutical composition prepared through the production method will be described by way of examples.

<Preparation Example 1> Preparation of Gynostrum extract

Gynecnospermia extract was prepared by mixing 100 parts by weight of Gynecnospermia powder prepared by drying and pulverizing Gynecdotes with 1000 parts by weight of ethanol with a mass concentration of 70% and solvent extracting it at a temperature of 22°C for 24 hours.

<Preparation Example 2> Cell culture

Mouse macrophage cell line RAW 264.7 cells were prepared and cultured in RPMI-1640 medium (Hyclone) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. The cells were cultured in an incubator under conditions of 37°C, 95% humidity, and 5% CO ₂ and subcultured with new culture medium every 2 to 3 days.

<Example 1> Confirmation of cytotoxicity

The RAW 264.7 cells prepared through Preparation Example 2 were seeded (1×10 ⁵ /well) in a 96 well cell plate, and the concentration of the Gynostrum extract prepared through Preparation Example 1 ranged from 1 μg/mL to 200 μg/mL. After treating each star for 24 hours, 10 ㎕ of MTS reagent was added and reacted for 2 hours. The absorbance was measured at a wavelength of 490 nm and is shown in Figures 1 and 2 below.

As shown in Figure 1 below, when confirmed with the MTS reagent, there was no cytotoxicity up to 50㎍/mL, but as shown in Figure 2 below, as a result of observing the cells under a microscope 24 hours after treatment with the Gynostrum extract at each concentration, 50 Cytotoxicity was observed at ㎍/mL.

<Example 2> Confirmation of efficacy in preventing influenza virus infection

1) Confirmation of antiviral efficacy using a fluorescence microscope

12 hours after treating Raw 264.7 cells cultured through Preparation Example 2 with 10 μg/mL and 20 μg/mL of the Gynostrum extract prepared in Preparation Example 1, influenza with a fluorescent protein (green fluorescence protein; gfp) attached After infection with the virus (PR8-GFP virus) at 10 MOI and further cultured for 12 or 24 hours, replication of VSV-gfp virus within cells was observed through gfp fluorescence expression, as shown in Figure 3 below. .(1000U/ml of IFN-β was used as a positive control.)

As shown in Figure 3 below, when infected with an influenza virus after treatment with a Gynostrum extract, it can be seen that the fluorescence displayed as the virus replicates decreases.

2) Confirmation of inhibition of CPE (cytopathic effect) caused by influenza virus

When the influenza virus replicates and proliferates, cells die and the CPE phenomenon occurs. Therefore, a CCK-8 assay was performed to confirm whether the Gynecnospermia extract inhibits CPE against influenza virus and increases cell survival rate.

Specifically, cells pretreated and cultured with Gynostrum extract in the same manner as above were infected with the virus, and 24 hours later, CCK-8 solution was added and reacted for 2 hours, and the color development results are shown in Figure 4 below. In addition, the supernatant was collected, the absorbance was measured at a wavelength of 450 nm, and the survival rate of the cells was calculated as a percentage relative to the medium, which is shown in Figure 5 below.

As shown in Figure 5 below, it can be seen that when infected with influenza virus after treating the Gynostrum extract prepared through Preparation Example 1 of the present invention, cell survival increases in a concentration-dependent manner.

Therefore, it was confirmed that the Gynecnospermia extract exhibits antiviral efficacy by inhibiting CPE caused by replication of the influenza virus.

<Example 3> Confirmation of efficacy in inhibiting influenza virus infection and proliferation

1) Confirmation of antiviral efficacy using a fluorescence microscope

Raw 264.7 cells cultured in Preparation Example 2 were simultaneously infected with 10 or 20 ㎍/mL of the Gynostrum extract prepared in Preparation Example 1 and influenza virus (PR8-GFP virus), and GFP fluorescence was photographed 24 hours later, as shown below. It is shown in Figure 6.

As shown in Figure 6 below, it can be seen that GFP fluorescence is significantly reduced in the presence of the Gynostemma extract prepared through Preparation Example 1.

2) Confirmation of antiviral efficacy through flow cytometry

Figure 7 shows that the cells identified in Figure 6 were collected by centrifugation, fixed with formalin solution with a mass concentration of 3.7%, resuspended in PBS (Phosphate buffered saline) solution, and analyzed for fluorescence using a flow cytometer (FACS, Fluorescence activated cell sorter). This is a histogram result analyzing the level of expression, and Figure 8 shows the results of numerical comparison and analysis of the fluorescence values obtained using a flow cytometer.

As shown in Figure 8, it can be seen that the Gynostrum extract prepared through Preparation Example 1 of the present invention has excellent efficacy in inhibiting infection and proliferation of influenza viruses.

3) Confirmation of efficacy in suppressing influenza virus protein expression through Western blot analysis

RAW 264.7 cells treated simultaneously with the Gynostrum extract and virus were collected by centrifugation in the same manner as was performed in the confirmation of antiviral efficacy using a fluorescence microscope in Example 2, the supernatant was discarded, the cells were disrupted, and the cells were suspended in RIPA buffer. The protein was quantified and 30㎍ was electrophoresed using SDS-PAGE and then transferred to a PVDF membrane.

As shown in Figure 9 below, as a result of confirmation using antibodies against the influenza proteins HA, PA, and M2, it was found that the Gynostrum extract prepared through Preparation Example 1 of the present invention inhibited the expression of viral proteins.

4) Confirmation of efficacy in suppressing influenza virus protein expression through fluorescence immunoassay (Immuno fluorescence technique)

After infection with the Gynostrum extract and influenza virus prepared in Preparation Example 1 above for 24 hours, the cells were washed with PBS and fixed in a methanol solution with a mass concentration of 100% for 5 minutes and in a 3.7% formalin solution for 10 minutes. Blocked for 1 hour in a PBS solution containing 1% BSA and 0.05% tween-20, reacted for 1 hour with primary antibodies against viral proteins HA, M2, NA, NP, NS1, and PA, washed three times with PBS, and Alexa588 This reacted with the bound secondary antibody (Red fluorescence). After washing three times with PBS, DAPI solution was added to stain the nuclei (blue fluorescence), and then merged under a fluorescence microscope to confirm the two colors of fluorescence by overlapping them.

As a result, as shown in FIG. 10 below, when influenza virus was infected in the presence of the Gynostrum extract prepared through Preparation Example 1 of the present invention, the expression of all viral proteins was suppressed, which indicates that the Gynostrum extract inhibits infection with the influenza virus. This result proves that it provides antiviral efficacy.

5) Promotion of anti-viral cytokine expression by qPCR

RAW264.7 cells were treated with Gypsum extract and PR8-GFP virus. Anti-viral cytokine mRNA was analyzed by qPCR and is shown in Figures 11 to 13 below. At this time, the primers used in qPCR analysis are shown in Table 1 below.

<Table 1>

As shown in Figures 11 to 13 below, there was no significant difference in IL-1β mRNA expression compared to IL-1b expression in MOCK treatment and PR8-GFP virus treatment. However, when treated with 10 and 20 ug/ml of Gynostrum extract, expression of 469.21 and 270.96 folds was induced (Figure 11). For TNF-α, expression of 4 and 5.79 folds was also induced upon treatment with 10 and 20 ug/ml of Gynostrum extract (Figure 12). In addition, IL-6 expression was induced at 133.27 and 273.52 folds upon treatment with 10 and 20 ug/ml of Gypsum extract, respectively (Figure 13).

Therefore, the antiviral pharmaceutical composition and health functional food composition containing the Gynectarsus extract as an active ingredient according to the present invention not only exhibit excellent antiviral effects, but also have low cytotoxicity, so they can be safely used even when taken for a long period of time for preventive purposes. there is.

Claims (11)

An antiviral pharmaceutical composition containing a Gynecnospermia extract as an active ingredient, characterized in that it contains a Gynecnospermia extract as an active ingredient.
In claim 1,
The antiviral pharmaceutical composition containing the Gynecnospermia extract as an active ingredient is an antiviral pharmaceutical composition containing the Gynecnospermia extract as an active ingredient, characterized in that it contains 5 to 40 μg/mL of the Gynecnospermia extract.
In claim 2,
An antiviral pharmaceutical composition containing the Gynecnospermia extract as an active ingredient, characterized in that the extract is contained in an amount of 10 to 20 μg/mL.
In claim 1,
An antiviral pharmaceutical composition containing the Gynostrum extract as an active ingredient, characterized in that the Gynecnospermia extract induces the antiviral factors TNF-α, IL-1β, and IL-6.
In claim 1,
The ginseng extract is characterized in that it is prepared by mixing 100 parts by weight of ginseng powder with 500 to 1,500 parts by weight of a solvent consisting of at least one selected from the group consisting of water and alcohols having 1 to 4 carbon atoms and extracting at room temperature for 20 to 30 hours. An antiviral pharmaceutical composition containing as an active ingredient the extract of Gynostrum.
In claim 5,
An antiviral pharmaceutical composition containing Gynecnospermia extract as an active ingredient, characterized in that the Gynecnospermia extract is prepared by mixing 100 parts by weight of Gynecnospermia powder with 1000 parts by weight of ethanol and extracting at room temperature for 24 hours.
In claim 1,
An antiviral pharmaceutical composition containing a Gynecnophyllum extract as an active ingredient, wherein the virus is an influenza virus.
In claim 1,
An antiviral pharmaceutical composition containing the Gynecnospermia extract as an active ingredient, characterized in that it further contains a pharmaceutically acceptable carrier, excipient, or diluent.
In claim 1,
The antiviral pharmaceutical composition containing the Gynecnocodon extract as an active ingredient is characterized by having a dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspension emulsions, syrups, aerosols, topical preparations, suppositories, and injections. An antiviral pharmaceutical composition containing Gynostrum extract as an active ingredient.
An antiviral health functional food composition containing Gynecnospermia extract as an active ingredient, characterized in that it contains Gynecnospermia extract as an active ingredient.
In claim 10,
The health functional food is an antiviral health functional food composition containing ginseng extract as an active ingredient, characterized in that it has one or more formulations selected from the group consisting of powder, granule, tablet, capsule, candy, chewing gum, jelly and beverage.