KR20240013998A - A composition for preventing and treating COVID-19 omicron variant virus (SARS-CoV-2 omicron variant virus) infection containing active ingredients isolated from Elaeocarpus sylvestris extract - Google Patents

Abstract

The present invention uses geraniin and pentagalloyl glucose (1,2,3,4,6-Penta- O- galloyl-β-D-glucose; PGG), which are compounds isolated from dampalsu extract, as active ingredients. It relates to a composition for improving, preventing or treating COVID-19 omicron mutant virus infection, etc., comprising: the compounds of the present invention are ingredients derived from natural plants, have little or no side effects, and contain S of the COVID-19 omicron mutant virus spike protein. By binding strongly to the RBD, it can significantly reduce the infection of host cells with the COVID-19 omicron mutant virus, so it is expected to be useful as a preventive or therapeutic agent for infection by the COVID-19 omicron mutant virus.

Description

Composition for preventing and treating COVID-19 omicron variant virus (SARS-CoV) infection containing active ingredients isolated from dampalsu extract {A composition for preventing and treating COVID-19 omicron variant virus (SARS-CoV) -2 omicron variant virus) infection containing active ingredients isolated from Elaeocarpus sylvestris extract}

The present invention uses geraniin and pentagalloyl glucose (1,2,3,4,6-Penta- O -galloyl-β-D-glucose; PGG), which are compounds isolated from dampal tree extract, as active ingredients. It relates to a composition for preventing, improving or treating COVID-19 omicron mutant virus infection, etc.

Severe acute respiratory syndrome coronavirus type 2, the cause of coronavirus disease-19 (Coronavirus disease-19; COVID-19; COVID-19, hereinafter referred to as COVID-19) that occurred in Wuhan, China in December 2019. 2; SARS-CoV-2) is a virus with RNA nucleic acid and is known to have a higher mutation rate than viruses with DNA nucleic acid. Since the start of the global epidemic, more than 10 SARS-CoV-2 mutant viruses (alpha, beta, gamma, delta, etc.) were confirmed.

Meanwhile, the 'SARS-CoV-2 omicron mutant virus (hereinafter referred to as COVID-19 omicron mutant virus)', which was first identified in South Africa in November 2021, is a mutant virus previously identified as the GR type, B.1.1.529 lineage. It was confirmed to have more mutations, and the World Health Organization (WHO) held an emergency meeting of the Technical Advisory Group on Virus Evolution (TAG-VE) on November 26, 2021 to determine the characteristics of the mutant virus. After evaluation, it was named 'Omicron', the 15th letter of the Greek alphabet, and classified as a variant of concern (VOC).

The COVID-19 omicron mutant virus is found to have more than 30 amino acid mutations in the spike protein (Spike Glycoprotein, used when the virus binds to host cell receptors), which is about twice as many as the delta mutation, in which 16 amino acid mutations were confirmed. It was confirmed that 15 of these are present in the spike receptor-binding domain (S RBD) protein region, and 15 of them are present in the previously confirmed mutant viruses (1 alpha, 3 beta, 3 gamma, and delta). It was confirmed that there were significantly more mutations in the S RBD protein region compared to 2). This means that there is a high possibility that there will be differences in protein structure and antigenicity from existing mutant viruses.

In addition, mutations (K417N, T478K, E484A, N501Y, D614G) at the same positions as the amino acid mutation sites identified in alpha, beta, gamma, and delta mutations were confirmed to exist, acquired through previous SARS-CoV-2 infection. It is expected to have a high possibility of evading the immune response generated by natural immunity and vaccination and to be highly contagious, and accordingly, the risk of reinfection and the speed of transmission are expected to increase compared to other major mutant viruses.

As of January 2022, the COVID-19 Omicron mutant virus has a high transmissibility and is spreading globally, and the spread rate has been shown to be about 2.5 times faster than the delta mutant. In Korea, the number of infected people exceeded 1,000 within a month of the introduction of the COVID-19 Omicron mutant virus, and as of January 24, 2022, the detection rate of the COVID-19 Omicron mutant virus was 50.3%, making it the dominant species. In addition, in the United States, 79% of those infected with the COVID-19 Omicron mutant virus were confirmed to be breakthrough infections (infected despite being vaccinated), and in Korea, half of the confirmed COVID-19 cases were reported to be breakthrough infections.

Accordingly, there is a need for a treatment targeting only the COVID-19 omicron mutant virus, separate from the coronavirus treatment targeting SARS-CoV-2 or previously identified mutant viruses (alpha, gamma, etc.).

Kannan S, Shaik Syed Ali P, Sheeza A. Omicron (B.1.1.529) - variant of concern - molecular profile and epidemiology: a mini review. Eur Rev Med Pharmacol Sci. 2021 Dec;25(24):8019-8022.

The present inventors conducted extensive research to develop a treatment for the COVID-19 omicron mutant virus, which has a high transmissibility and is spreading worldwide, and as a result, geranin and PGG isolated from dampalsu extract were found to be effective against the COVID-19 omicron mutant virus. The present invention was completed by confirming that infection of host cells with the COVID-19 omicron mutant virus can be effectively inhibited by binding to the S RBD.

Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating COVID-19 omicron mutant virus infection containing a compound of the following formula 1 or 2, or a pharmaceutically acceptable salt thereof, as an active ingredient.

[Formula 1]

[Formula 2]

Another object of the present invention is to provide a food composition for preventing or improving COVID-19 omicron mutant virus infection containing the compound of Formula 1 or 2, or a foodologically acceptable salt thereof, as an active ingredient.

Another object of the present invention is to provide a quasi-drug composition for preventing or improving COVID-19 omicron mutant virus infection containing the compound of Formula 1 or 2, or a pharmaceutically acceptable salt thereof, as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating COVID-19 omicron mutant virus infection comprising a compound of formula 1 or 2 below, or a pharmaceutically acceptable salt thereof, as an active ingredient. provides.

[Formula 1]

[Formula 2]

In addition, the present invention provides a food composition for preventing or improving COVID-19 omicron mutant virus infection, comprising the compound of Formula 1 or 2, or a foodologically acceptable salt thereof, as an active ingredient.

In addition, the present invention provides a quasi-drug composition for preventing or improving COVID-19 omicron mutant virus infection, comprising the compound of Formula 1 or 2, or a pharmaceutically acceptable salt thereof, as an active ingredient.

In one embodiment of the present invention, the compound may be isolated from dampalsu extract, but is not limited thereto.

In another embodiment of the present invention, the dampal tree extract may be a dampal tree leaf extract, but is not limited thereto.

In another embodiment of the present invention, the dampal water extract is water, alcohol having 1 to 6 carbon atoms, acetone, ether, benzene, chloroform, and ethyl acetate. ), methylene chloride, hexane, cyclohexane, petroleum ether, subcritical fluid, and supercritical fluid. It is not limited to this.

In another embodiment of the present invention, the compound may bind to the S RBD protein of the COVID-19 omicron mutant virus, but is not limited thereto.

In another embodiment of the present invention, the S RBD protein may be represented by the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

As another embodiment of the present invention, the food composition may be a health functional food composition, but is not limited thereto.

In addition, the present invention provides a method for preventing COVID-19 omicron mutant virus infection, comprising administering a composition containing the compound of Formula 1 or 2, or a pharmaceutically acceptable salt thereof, as an active ingredient to an individual in need thereof. Or provide a treatment method.

In addition, the present invention provides the use of a composition containing the compound of Formula 1 or 2, or a pharmaceutically acceptable salt thereof as an active ingredient, for preventing or treating COVID-19 omicron mutant virus infection.

In addition, the present invention provides the use of the compound of Formula 1 or 2, or a pharmaceutically acceptable salt thereof, for preparing a medicament for preventing or treating COVID-19 omicron mutant virus infection.

The compounds of the present invention are ingredients derived from natural plants, have little or no side effects, and can inhibit infection of host cells by the COVID-19 omicron mutant virus by strongly binding to the S RBD protein of the COVID-19 omicron mutant virus. It is expected that it can be useful as a preventative or therapeutic agent for COVID-19 omicron mutant virus infection.

Figure 1 shows the three-dimensional S RBD protein structure (left) of the COVID-19 virus (SARS-Cov-2 virus) modeled using SWISS-MODEL and the molecular docking results of the three-dimensional S RBD protein structure and geranin or PGG. This is a diagram (the part marked with a yellow box indicates the site where S RBD protein and geranin or PGG bind).
Figure 2 is a diagram showing the three-dimensional S RBD protein structure of the COVID-19 Omicron mutant virus (SARS-Cov-2 Omicron B.1.1.529) modeled using SWISS-MODEL.
Figure 3 is a diagram showing the three-dimensional S RBD protein structure of the COVID-19 Omicron mutant virus (SARS-Cov-2 Omicron) and the molecular docking results of geranin or PGG (the part marked with a yellow box represents the S RBD protein and geranin). Indicates the site where nin or PGG binds).
Figure 4 is a diagram showing the three-dimensional S RBD protein structure of the COVID-19 Stealth Omicron Mutant Virus (SARS-Cov-2 Omicron BA.2) modeled using SWISS-MODEL.
Figure 5 is a diagram showing representative blind molecular docking results of the S RBD protein of the modeled COVID-19 stealth omicron mutant virus and geranin or PGG (the part marked with a red box is where geranin or PGG is bound to in the S RBD protein). indicates the area).
Figure 6 is a diagram showing the region where geranin or PGG binds in the S RBD protein of the modeled COVID-19 stealth omicron mutant virus.
Figure 7 is a diagram showing the results of confirming the binding inhibition rate of geranin and PGG to the S RBD of the COVID-19 virus and its variant viruses (alpha, beta, delta, and omicron).
Figure 8 is a diagram showing the results confirming the antiviral effect of geranin on the COVID-19 omicron mutant virus (blue ○ indicates infection inhibition rate, red □ indicates cell survival rate).
Figure 9 is a diagram showing a test report confirming the content of geranin, an indicator component of dampalsu extract.

The terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part is said to “include” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.

All technical terms used in the present invention, unless otherwise defined, are used with the same meaning as commonly understood by a person skilled in the art in the field related to the present invention. In addition, preferred methods and samples are described in this specification, but similar or equivalent methods are also included in the scope of the present invention. The contents of all publications incorporated by reference herein are hereby incorporated by reference.

The present inventors have developed a highly transmissible COVID-19 omicron mutant virus that is spreading globally in a situation where treatments and vaccines with proven antiviral effects have not been developed against SARS-CoV-2, which has various mutations. As a result of intensive research to develop a treatment for The present invention was completed by confirming that infection of host cells with the COVID-19 omicron mutant virus can be effectively inhibited.

In one example of the present invention, geranin and PGG were isolated from dampalsu extract (see Example Ⅰ).

In another embodiment of the present invention, the COVID-19 omicron mutant virus and COVID-19 stealth virus are difficult to model due to significant structural differences due to significantly more mutations occurring compared to the previously identified COVID-19 virus or its mutant virus. The S RBD structure of the micron mutant virus was modeled, and it was confirmed that geranin or PGG exhibits high binding affinity to the S RBD protein of the COVID-19 omicron mutant virus and the COVID-19 stealth omicron mutant virus (see Example II) .

In another embodiment of the present invention, geranin and PGG have significant RBD-ACE2 binding compared to the COVID-19 virus and its mutant viruses (alpha, beta, delta), especially to the S RBD of the COVID-19 omicron mutant virus. It was confirmed that it exhibited an inhibition rate (see Example III).

In another embodiment of the present invention, compared to chloroquine, lopinavir, and remdesivir, which are used as treatments for the COVID-19 virus, geranine is effective against the COVID-19 omicron mutant virus. It was confirmed that the antiviral effect was superior (see Example IV).

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating COVID-19 omicron mutant virus infection, comprising a compound of formula 1 or 2 below, or a pharmaceutically acceptable salt thereof, as an active ingredient.

[Formula 1]

[Formula 2]

The compound represented by Formula 1 is named geraniin (GE), is known to inhibit apoptosis induced by radiation, and is contained in high amounts in Dampalsu extract.

The compound represented by Formula 2 is pentagalloyl glucose (1,2,3,4,6-Penta- O -galloyl-β-D-glucose; PGG) is a pentagallic acid ester of glucose and a precursor of gallotannin and ellagitannin. It is known to be synthesized using 1- O -galloyl-beta-D-glucose and 1,2,3,6-tetrakis- O -galloyl-beta-D-glucose. The functions of PGG include inhibiting the formation of blood vessels in vascular endothelial cells, inhibiting the differentiation of adipocytes, inhibiting signal transduction by IL-4 stimulation, intercellular signaling, inhibiting angiogenesis, and protecting against radiation. It has been reported.

The present invention may include not only the compound represented by Formula 1 or Formula 2, or a racemate thereof, or a pharmaceutically acceptable salt thereof, but also all possible solvates that can be prepared therefrom, and the Both active ingredients and metabolites of geranine may be included.

The active ingredients and geranin metabolites of the dampalsu extract include, for example, corilagin (Luger et al. 1998), ellagic acid (Luger et al. 1998), gallic acid (Luger et al. 1998), punigluconin, and syzyginin B. , rutin, pentagalloyl glucose, emblicanin A, ursolic acid, quercetin, dihydromyricetin, taxifolin, aromadendrin and kaempferol, etc., but is not limited thereto.

The corilagin is produced by partial hydrolysis of geranine, and ellagic acid and gallic acid are metabolites produced by complete hydrolysis (Hodgson et al. 2000; Ito 2011), but are not limited to this and are involved in the metabolism of geranine. Anything created in the process can be included.

In the present invention, “metabolite” refers to a substance involved in metabolism or produced during the metabolism process.

As used herein, the term “pharmaceutically acceptable salt” includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases.

Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid. , benzoic acid, malonic acid, gluconic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Acid addition salts can be prepared by conventional methods, for example, by dissolving the compound in an excessive amount of aqueous acid and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone, or acetonitrile. It can also be prepared by heating equimolar amounts of the compound and an acid or alcohol in water and then evaporating the mixture to dryness, or suction filtering the precipitated salt.

Salts derived from suitable bases may include, but are not limited to, alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium. The alkali metal or alkaline earth metal salt can be obtained, for example, by dissolving the compound in an excessive amount of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and then evaporating and drying the filtrate. At this time, it is particularly pharmaceutically suitable to prepare sodium, potassium or calcium salts as metal salts, and the corresponding silver salts can be obtained by reacting an alkali metal or alkaline earth metal salt with an appropriate silver salt (eg, silver nitrate).

The content of the compound of Formula 1 or 2, or a pharmaceutically acceptable salt thereof, in the composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the degree of progression of the symptoms, the patient's condition, etc., for example, based on the total weight of the composition. It may be 0.0001 to 99.9% by weight, or 0.001 to 50% by weight, but is not limited thereto. The content ratio is a value based on the dry amount with the solvent removed.

In the present invention, the compound may be isolated from a dampalsu extract, but is not limited thereto, and may be isolated from other natural products, obtained through organic synthesis, or a commercially available material.

“ Elaeocarpus sylvestris ” of the present invention is a plant of the Elaeocarpus sylvestris family and is a deciduous broad-leaved tree that mainly grows in Jeju Island, Japan, Taiwan, etc. in Korea. It grows up to 20 m, and the leaves are long oval or lanceolate and 6-12 inches long. cm and has a width of 1.5-3 cm. The roots of the Dampalsu tree have been called Sanduyeong in Oriental medicine since ancient times and have been used to treat diseases such as fever, diarrhea, and cough.

The dampal water of the present invention can be used as is without damaging its original form, or can be used after performing a pretreatment process in consideration of the process speed and process (manufacturing) efficiency intended by those skilled in the art. The pretreatment process includes, for example, conventional It can go through steps such as screening, washing, cutting, powdering, and drying.

The dampalsu extract of the present invention may be obtained from the whole plant or part of the plant, for example, from a part containing one or more selected from the group consisting of stems, roots, leaves, fruits, and flowers, but according to the present invention According to one embodiment, the dampal tree extract may be a dampal tree leaf extract.

In addition, in the present invention, the dampalsu extract may contain geranin as an indicator component, and in this case, the content of geranin as an indicator component may be characterized as containing 9.8% or more on a dry weight basis.

In the present invention, “indicator component” refers to a component determined for the purpose of quality control among chemically identified components contained in raw materials, and in the present invention, “active ingredient” refers to a component that alone exhibits the desired activity or is itself means an ingredient that can exhibit the desired activity together with an inactive carrier, etc.

The geranin, which is an active ingredient of the composition according to the present invention, is an indicator component of the dampal water extract and is characterized in that it is contained in a high content in the dampal water extract. The dampal water extract particularly prevents and improves the COVID-19 omicron mutant virus. , or can be useful for treatment.

The dampalsu extract of the present invention can be extracted by a known natural product extraction method. For example, it may be extracted with one or more solvents selected from the group consisting of water, organic solvents having 1 to 6 carbon atoms, and subcritical or supercritical fluids. The organic solvent having 1 to 6 carbon atoms is alcohol, acetone, ether, benzene, chloroform, ethyl acetate, and methylene chloride. It may be one or more selected from the group consisting of (methylene chloride), hexane, cyclohexane, and petroleum ether, but is not limited thereto.

The dampalsu extract of the present invention may preferably be extracted with ethanol, but is not limited thereto. Conventional extraction methods such as cold immersion, warm immersion, and heating can be used using the above solvent.

The concentration of the ethanol is not limited, but for example, 30 to 100%, 40 to 100%, 50 to 100%, 60 to 100%, 70 to 100%, 80 to 100%, 90 to 100%, 95 to 100%. It may be 100% or 50% ethanol extract.

The term “COVID-19 omicron mutant virus infection” used in the present invention refers to COVID-19 omicron mutant virus (B.1.1.529 lineage), which is one of the COVID-19 mutant viruses, and its submutated viruses (e.g., It refers to a disease caused by infection with the COVID-19 stealth omicron mutant virus.

The above “COVID-19 Stealth Omicron Mutant Virus (BA.2 Lineage)” refers to a sub-mutant virus of the COVID-19 Omicron Mutant Virus.

Coronaviruses, including the SARS-CoV-2 omicron mutant virus, have the S RBD portion of the spike protein on the envelope binding to the angiotensin-converting enzyme 2 (ACE2) receptor of the host cell. It is known to be infected inside the living body, and accordingly, there have been many attempts to prevent or treat coronavirus infection by targeting the S RBD portion of the virus's spike protein and interfering with its binding to host cells.

According to one embodiment of the present invention, the compound is S of the COVID-19 omicron mutant virus, which has many structural differences from the previously identified COVID-19 virus or its mutant virus due to 15 mutations occurring in the S RBD protein. GLY129, TYR131, LEU174, ARG175, and/or ARG10, CYS73, LYS210 amino acid residues of RBD proteins (for PGG); Alternatively, it binds strongly through hydrogen bonds with the CYS61 amino acid residue (in the case of geranin, two or more hydrogen bonds), thereby preventing the binding of the S RBD protein of the COVID-19 omicron mutant virus to the ACE2 receptor of the host cell, causing COVID-19. It can prevent omicron mutant viruses from infecting the body.

In addition, according to one embodiment of the present invention, the compound is THR430, PHE515, ASN360, ASN388, and/or ASP389 of the S RBD protein of COVID-19 stealth omicron mutant virus, which is a submutated COVID-19 omicron mutant virus. By strongly binding to amino acid residues through hydrogen bonding, it prevents the binding of the S RBD protein of the COVID-19 stealth omicron mutant virus to the ACE2 receptor of the host cell, preventing the COVID-19 omicron mutant virus from infecting the body. You can.

In the present invention, the S RBD protein of the COVID-19 omicron mutant virus may be represented by the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 2, but is not limited thereto, and variants of the amino acid sequence are within the scope of the present invention. may be included. The polypeptide having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 of the present invention is a functional equivalent of the nucleic acid molecule constituting it, for example, some amino acid sequences of the polypeptide are deleted, substituted, or inserted ( It is a concept that includes variants that have been modified by insertion but can have the same functional effect as the polypeptide. Specifically, the S RBD of the COVID-19 omicron mutant virus contains at least 70%, more preferably at least 80%, and even more preferably at least 90% of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, respectively. Most preferably, it may include an amino acid sequence having 95% or more sequence homology. For example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85. %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology. It includes the S RBD of the COVID-19 omicron mutant virus. The “% sequence homology” for a polypeptide is determined by comparing a comparison region with two optimally aligned sequences, where a portion of the polypeptide sequence in the comparison region is a reference sequence (including additions or deletions) for the optimal alignment of the two sequences. may contain additions or deletions (i.e. gaps) compared to those that do not.

In the present invention, the compound is the 7th to 13th amino acid, 7th to 10th amino acid, 10th to 13th amino acid, and 58th amino acid of the S RBD protein of the COVID-19 omicron mutant virus represented by SEQ ID NO: 1. 64th amino acid, 58th to 61st amino acid, 61st to 64th amino acid, 70th to 76th amino acid, 70th to 73rd amino acid, 73rd to 76th amino acid, 126th to 132nd amino acid, 126th to 129th amino acid amino acid, 129th to 131st amino acid, 131st to 132nd amino acid, 171st to 177th amino acid, 171st to 174th amino acid, 174th to 175th amino acid, 175th to 177th amino acid, 207th to 213th amino acid Amino acids, amino acids 207 to 210, amino acids 210 to 213, amino acids 10, amino acids 61, amino acids 73, amino acids 129, amino acids 131, amino acids 174, amino acids 175, and/or amino acids 210 It may specifically bind to the region indicated by , but is not limited thereto.

In addition, the compound contains the 354th to 363rd amino acids, 354th to 360th amino acids, 360th to 363rd amino acids, and 357th amino acids of the S RBD protein of the COVID-19 stealth omicron mutant virus shown in SEQ ID NO: 2. 360th amino acid, 382nd to 391st amino acid, 382nd to 388th amino acid, 388th to 391st amino acid, 385th to 388th amino acid, 388th to 389th amino acid, 389th to 391st amino acid, 422nd to 431 amino acid, 425th to 430th amino acid, 430th to 431st amino acid, 513th to 522nd amino acid, 513th to 515th amino acid, 515th to 522nd amino acid, 515th to 519th amino acid, 360th amino acid, 388 It may specifically bind to the region represented by the 389th amino acid, 389th amino acid, 430th amino acid, and/or 515th amino acid, but is not limited thereto.

In the present invention, “pharmaceutical composition” means manufactured for the purpose of preventing or treating disease, and can be formulated and used in various forms according to conventional methods. For example, it can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, etc., and can be formulated and used in the form of external preparations, suppositories, and sterile injection solutions.

The pharmaceutical composition according to the present invention may further include appropriate carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. The excipient may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, humectants, film-coating materials, and controlled-release additives.

The pharmaceutical composition according to the present invention can be prepared as powder, granules, sustained-release granules, enteric-coated granules, solutions, eye drops, ellipsis, emulsions, suspensions, spirits, troches, perfumes, and limonadese according to conventional methods. , tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric-coated capsules, pills, tinctures, soft extracts, dry extracts, liquid extracts, injections, capsules, perfusate, It can be formulated and used in the form of external preparations such as warning agents, lotions, paste preparations, sprays, inhalants, patches, sterilized injection solutions, or aerosols, and the external preparations include creams, gels, patches, sprays, ointments, and warning agents. , it may have a dosage form such as lotion, liniment, pasta, or cataplasma.

Carriers, excipients, and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, and calcium. These include phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Additives to tablets, powders, granules, capsules, pills, and troches according to the present invention include corn starch, potato starch, wheat starch, lactose, white sugar, glucose, fructose, di-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, and phosphoric acid. Calcium monohydrogen, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethyl. Excipients such as cellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate, carboxymethyl cellulose, calcium carboxymethyl cellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethyl cellulose, sodium methyl cellulose, methyl cellulose, microcrystalline cellulose, dextrin. , hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc. binders can be used, Hydroxypropyl methyl cellulose, corn starch, agar powder, methyl cellulose, bentonite, hydroxypropyl starch, sodium carboxymethyl cellulose, sodium alginate, calcium carboxymethyl cellulose, calcium citrate, sodium lauryl sulfate, silicic acid anhydride, 1-hydroxy Propylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, Disintegrants such as amylopectin, pectin, sodium polyphosphate, ethyl cellulose, white sugar, magnesium aluminum silicate, di-sorbitol solution, light anhydrous silicic acid; Calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium, kaolin, petrolatum, sodium stearate, cacao fat, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogen. Added soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, Macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acids, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, Lubricants such as starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid; can be used.

Additives to the liquid according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, etc. can be used.

A solution of white sugar, other sugars, or sweeteners, etc. may be used in the syrup according to the present invention, and if necessary, flavoring agents, colorants, preservatives, stabilizers, suspending agents, emulsifiers, thickening agents, etc. may be used.

Purified water can be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. can be used as needed.

Suspensions according to the present invention include acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, HPMC 2910, etc. Topics may be used, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

Injections according to the present invention include distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oil - sesame oil. solvents such as cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristic acid, and benzene benzoate; Solubilizers such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, Tween, nicotinic acid amide, hexamine, and dimethylacetamide; Weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and buffering agents such as gums; Isotonic agents such as sodium chloride; Stabilizers such as sodium bisulfite (NaHSO ₃ ) carbon dioxide gas, sodium metabisulfite (Na ₂ S ₂ O ₅ ), sodium sulfite (Na ₂ SO ₃ ), nitrogen gas (N ₂ ), and ethylenediaminetetraacetic acid; Antioxidants such as sodium bisulfite 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, and acetone sodium bisulfite; Analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; It may contain suspending agents such as CM sodium, sodium alginate, Tween 80, and aluminum monostearate.

Suppositories according to the present invention include cacao oil, lanolin, witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, lecithin, Lanet wax, glycerol monostearate, Tween or Span, Imhausen, Monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydrocote SP, S-70-XXA, S-70-XX75(S-70-XX95), Hydro Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Massupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), suppositories type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecobi (W, R, S, M, Fs), Tegestor triglyceride base (TG-95, MA, 57) and The same mechanism can be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include the extract with at least one excipient, such as starch, calcium carbonate, or sucrose. ) or prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate talc are also used.

Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, drug activity, and It can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the medical field.

The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art to which the present invention pertains.

The pharmaceutical composition of the present invention can be administered to an individual through various routes. All modes of administration are contemplated, including oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal injection. It can be administered by internal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, etc.

The pharmaceutical composition of the present invention is determined depending on the type of drug as the active ingredient along with various related factors such as the disease to be treated, the route of administration, the patient's age, gender, weight, and severity of the disease.

In addition, the present invention provides a method for preventing COVID-19 omicron mutant virus infection, comprising administering a composition containing the compound of Formula 1 or 2, or a pharmaceutically acceptable salt thereof, as an active ingredient to an individual in need thereof. Or provide a treatment method.

In addition, the present invention provides the use of a composition containing the compound of Formula 1 or 2, or a pharmaceutically acceptable salt thereof as an active ingredient, for preventing or treating COVID-19 omicron mutant virus infection.

In addition, the present invention provides the use of the compound of Formula 1 or 2, or a pharmaceutically acceptable salt thereof, for preparing a medicament for preventing or treating COVID-19 omicron mutant virus infection.

In the present invention, “individual” refers to a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses, cows, etc. refers to mammals of

In the present invention, “administration” means providing a given composition of the present invention to an individual by any appropriate method.

In the present invention, “prevention” refers to any action that suppresses or delays the onset of the desired disease, and “treatment” refers to the improvement or improvement of the desired disease and its associated metabolic abnormalities by administration of the pharmaceutical composition according to the present invention. It refers to all actions that are beneficially changed.

In addition, the present invention provides a food composition for preventing or improving COVID-19 omicron mutant virus infection, comprising the compound of Formula 1 or 2, or a foodologically acceptable salt thereof, as an active ingredient.

In the present invention, the term “foodologically acceptable salt” includes salts derived from foodologically acceptable organic acids, inorganic acids, or bases.

In the present invention, “improvement” means any action that reduces parameters related to the target disease, such as the degree of symptoms, by administering the composition according to the present invention.

When using the compound of formula 1 or 2 of the present invention, or a foodologically acceptable salt thereof, as a food additive, the compound of formula 1 or 2, or a foodologically acceptable salt thereof, may be added as is or added to other foods or food products. It can be used together with the ingredients and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). In general, when manufacturing a food or beverage, the compound of Formula 1 or 2 of the present invention, or a foodologically acceptable salt thereof, may be added in an amount of 15% by weight or less, or 10% by weight or less, based on the raw materials. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

There are no special restrictions on the types of foods above. Examples of foods to which the above substances can be added include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, These include alcoholic beverages and vitamin complexes, and include all health functional foods in the conventional sense.

The health drink composition according to the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, like conventional drinks. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, natural sweeteners such as thaumatin and stevia extract or synthetic sweeteners such as saccharin and aspartame can be used. The proportion of natural carbohydrates is generally about 0.01-0.20 g, or about 0.04-0.10 g per 100 mL of the composition of the present invention.

In addition to the above, the composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, It may contain carbonating agents used in carbonated drinks. Additionally, the composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages. These ingredients can be used independently or in combination. The ratio of these additives is not very important, but is generally selected in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention.

In the present invention, the food composition may be a health functional food composition.

In this specification, “health functional food” is the same term as food for special health use (FoSHU), and refers to food with high medical and medical effects that has been processed to efficiently exhibit bioregulatory functions in addition to supplying nutrients. This means that the food can be manufactured in various forms such as tablets, capsules, powders, granules, liquids, pills, etc. to achieve useful effects in preventing or improving obesity.

The health functional food of the present invention can be manufactured by a method commonly used in the art, and can be manufactured by adding raw materials and components commonly added in the art. In addition, unlike general drugs, it is made from food, so it has the advantage of not having any side effects that may occur when taking the drug for a long time, and it can be highly portable.

In addition, the present invention provides a quasi-drug composition for preventing or improving COVID-19 omicron mutant virus infection, comprising the compound of Formula 1 or 2, or a pharmaceutically acceptable salt thereof, as an active ingredient.

The above quasi-drugs are fibers, rubber products or similar products used for the purpose of treating, alleviating, treating or preventing diseases in humans or animals, have a weak effect on the human body or do not act directly on the human body, and are not instruments or machines. Similar items, products that fall under one of the preparations used for sterilization, insecticide and similar purposes to prevent infectious diseases, such as instruments used for the purpose of diagnosing, treating, mitigating, treating or preventing diseases in humans or animals; It refers to articles that are not machines or devices and that are used for the purpose of having a pharmacological effect on the structure and function of humans or animals, excluding those that are not instruments, machines, or devices.

The type or formulation of the quasi-drug composition of the present invention is not particularly limited, and may be formulated in various forms as quasi-drugs known in the art to exhibit antiviral activity. The above-formulated quasi-drugs include disinfectant cleaners, shower foam, garnish, wet wipes, detergent soap, hand wash, ointment, cream, lotion, spray, humidifier filler, mask, ointment, patch or filter filler, etc., and are not used in the usual sense. Includes all quasi-drugs.

When adding the compound of the present invention to a quasi-drug composition for the purpose of preventing or improving the COVID-19 omicron mutant virus infectious disease, the compound can be added as is or used together with other quasi-drugs or quasi-drug components, and can be prepared by conventional methods. It can be used appropriately. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use, and may include, for example, thickeners, stabilizers, solubilizers, vitamins, conventional auxiliaries such as pigments and flavorings, and carriers.

Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only for easier understanding of the present invention, and the content of the present invention is not limited by the following examples.

[Example]

Example I. Isolation of active ingredients of dampalsu extract

1. Preparation of dampalsu extract

The leaves of domestic Dampalsu were obtained from around Jeju Island, dried, and cut into pieces. Then, 100 kg of Dampalsu leaves were extracted with 2,000 L of 50% ethanol at 60°C for 16 hours. After filtration and concentration, they were dried to produce 28 kg of Dampalsu extract (yield 28%). got it

2. Isolation of geranin and PGG from dampalsu extract

10 g of the dampalsu extract prepared above was suspended in 50 mL of distilled water, and then sequentially fractionated into equal amounts of dimethyl chloride (MC) and normal butanol (n-BuOH), followed by concentration under reduced pressure to obtain a butanol fraction.

The obtained butanol fraction (3 g) was subjected to column chromatography (MeOH-Water 20:1 → 40:1 → 60:1 → 80:1, 300 mL each) on an ODS column (ϕ 4 × 7 cm), and TLC Based on the phase, it was divided into four subfractions (ESE-Bu-1~4). Of each obtained fraction, the third subfraction (ESE-Bu-3, 1.2 g) was re-fractionated by applying MeOH-water 1:2 conditions to ODS semi-preparative TLC, and the band visualized under 280 nm UV light was scraped. Thus, Compound 1 and Compound 2 were obtained. To confirm their chemical structures, each compound was dissolved in deuterium-substituted methanol (CD ₃ OD) and 600 MHz NMR was measured.

As a result, compound 1 in the butanol fraction of dampalsu extract was confirmed to be pentagalloyl glucose (1,2,3,4,6-Penta- O -galloyl-β-D-glucose; PGG), and compound 2 was geranin. (Geraniin).

Example II. in silico Analysis of the combination of COVID-19 omicron variant virus (B.1.1.529) with PGG and geranin using an analytical method

1. S RBD protein modeling of COVID-19 omicron mutant virus

1-1. modeling method

To construct the amino acid sequence of the S RBD of the COVID-19 omicron mutant virus, the amino acid sequence of the S RBD of the COVID-19 virus was used (GenBank Accession Number QHD43416.1). 34 mutant sequences of S RBD of COVID-19 omicron mutant virus (A67V, HV 69-70 deletion, T95I, GVY 142-144 deletion, Y145D, N211 deletion, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N , N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L 981F) of the COVID-19 virus The amino acid sequence of the S RBD of the COVID-19 omicron mutant virus was created by modifying the amino acid sequence of the S RBD. SWISS-MODEL ( https://swissmodel.expasy.org ; a web that models three-dimensional protein structures based on the template) by importing the amino acid sequence portion (319-541) of the S RBD of the produced COVID-19 omicron mutant virus Based service) was used to model the three-dimensional protein structure of the S RBD of the COVID-19 omicron mutant virus.

1-2. Modeling verification method

GMQE (Global Model Quality Estimation) and QMEAN (Qualitative Model Energy Analysis) were used to confirm whether the three-dimensional protein structure of the S RBD of the COVID-19 omicron mutant virus was properly modeled. GMQE represents a normalized accuracy index from 0 to 1, and QMEAN represents a qualitative index, and a score lower than -4.0 means poor quality.

Additionally, to verify the three-dimensional protein structure of the S RBD of the COVID-19 omicron mutant virus, G-factor and Ramachandran plot were calculated using PROCHECK (https://www.ebi.ac.uk/thorntonsrv/software/PROCHECK). did. G-factor, which is a value indicating the degree of structural abnormality, means that if the score is lower than -0.5, the structure is abnormal, and if it is lower than -1.0, it means that the structure is very abnormal. The Ramachandran plot, which is widely used in verifying three-dimensional protein structures, is a graph that shows the torsion angles (phi and psi) of amino acid residues. In the Ramachandran plot results, more than 90% of the amino acid residues in the protein structure entered are the most favored regions. ), the modeling quality is judged to be good.

1-3. Identification of S RBD protein of modeled COVID-19 omicron mutant virus

As a result of applying 34 mutant sequences based on the S RBD amino acid sequence of the COVID-19 virus, the S RBD protein of the COVID-19 omicron mutant virus with the amino acid sequence of SEQ ID NO. 1 was obtained.

In SWISS-MODEL, 1,073 templates similar to the amino acid sequence (SEQ ID NO: 1) of the S RBD protein of the COVID-19 omicron mutant virus were discovered in SMTL, and among the 1,073 templates, there was 61% sequence similarity with template 7LYQ.1.A. (sequence similarity), showed 94.17% sequence identity. The S RBD protein structure of the COVID-19 omicron mutant virus has a total of 669 amino acid residues, and the GMQE and QMEAN values were 0.81 and 0.74 ± 0.05, respectively. The G-factor values of the S RBD protein model of the COVID-19 omicron mutant virus obtained using PROCHECK were -0.38 (dihedrals), -0.08 (covalent), and -0.24 (overall). As a result of the Ramachandran plot, 87.7% of amino acid residues are located in the most favored regions ([A, B, L]), and 87.7% are located in additional allowed regions ([a, b, l, p]). Amino acid residues were found to be 11.8%.

From the above results, it was confirmed that the S RBD protein structure of the COVID-19 omicron mutant virus modeled using SWISS-MODEL was stereochemically stable and accurate.

2. Molecular Docking Analysis

Molecular docking analysis with geranin and PGG was performed using the modeled S RBD three-dimensional protein structure of the COVID-19 omicron mutant virus.

2-1. Molecular docking analysis method

Analyzing the binding affinity and binding structure between geranin or PGG and the S RBD protein of COVID-19 omicron mutant virus using AutoDock (The Scripts Research Institute, La Jolla, CA, USA), an open source molecular docking simulation software. did. The structures of PGG and geranin were converted to '.pdb' format using Open Babel (http://openbabel.org) software, and the structures of PGG and geranin in the converted format and S of COVID-19 omicron mutant virus were converted to '.pdb' format using Open Babel (http://openbabel.org) software. RBD proteins were loaded into AutoDock. After adding polar hydrogen molecules to geranin, PGG, and the S RBD protein of the COVID-19 omicron mutant virus, the Kollman charge was calculated for the S RBD protein of the COVID-19 omicron mutant virus, and the Gasteiger charge was calculated for geranin and PGG. Geranin, PGG, and S RBD proteins of COVID-19 omicron mutant virus were converted to '.pdbqt' (Protein Data Bank, Partial Charge (Q), and Atom Type (T)) format, which is the input format for molecular docking simulation. . Since the binding site of the S RBD of the COVID-19 omicron mutant virus is unknown, the entire protein was set up as a grid map and then blind docking, a molecular docking technique, was performed. After setting the center point of the grid map to the center of the protein, the grid size was set to 100 × 100 × 100 Å and the grid space was set to 0.750 Å. The algorithm used in the AutoDock molecular docking simulation used the standard Lamarkian genetic algorithm. After conducting blind docking simulations with a total number of runs of 50 and a number of n of 2, the lowest binding energy (the most stable) was selected. A binding structure with binding energy) was derived.

2-2. Molecular docking analysis of geranin and PGG against the S RBD protein of COVID-19 omicron mutant virus.

To analyze the binding mechanism of PGG and geranin to the S RBD protein of the COVID-19 omicron mutant virus, blind docking simulations using AutoDock were performed several times for each compound.

As a result of blind docking analysis of PGG against the S RBD protein of the COVID-19 omicron mutant virus, the average binding energy of PGG and the S RBD protein of the COVID-19 omicron mutant virus was -2.81 kcal/mol, and the representative blind docking result is It is shown in Figure 3.

As a result of blind docking analysis of geranin against the S RBD protein of the COVID-19 omicron mutant virus, the average binding energy between geranin and the S RBD protein of the COVID-19 omicron mutant virus was -12.35 kcal/mol, a representative blind docking The results are shown in Figure 3.

Meanwhile, as a result of blind docking analysis of the S RBD protein of geranin or PGG and the COVID-19 virus, the average binding energy of the S RBD protein of geranin and the COVID-19 virus was -9.43 kcal/mol, PGG and the COVID-19 virus The average binding energy of the S RBD protein was -3.10 kcal/mol, and a representative blind docking result for the S RBD protein of the COVID-19 virus is shown in Figure 1.

S RBD covid-19 COVID-19 Omicron mutation Geranine (kcal/mol) -9.43 -12.35 PGG (kcal/mol) -3.10 -2.81

3. Analysis of the combination of SARS-CoV-2 stealth omicron variant virus (BA.2) with geranin and PGG

3-1. Method of securing S RBD protein of COVID-19 stealth omicron variant virus (SARS-CoV-2 stealth omicron variant virus, BA.2)

The S RBD protein of the COVID-19 stealth omicron mutant virus used the 'PDB ID: 7ZF7' model, which is a model combined with Angiotensin-converting Enzyme 2 (ACE2), from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) website. The S RBD model of the stealth omicron mutant virus with the ACE2 protein removed was obtained in '.pdb' file format and used for molecular docking analysis.

The amino acid sequence of the S RBD protein of the obtained COVID-19 stealth omicron mutant virus is shown as SEQ ID NO: 2.

3-2. Molecular docking analysis of geranin and PGG against the S RBD protein of COVID-19 stealth omicron mutant virus.

Blind molecular docking of geranin or PGG to the S RBD protein of the COVID-19 stealth omicron mutant virus was performed in the same manner as the COVID-19 omicron mutant virus except for the method of securing the S RBD protein.

As a result, as shown in Figure 5, the average binding energy of geranin and the S RBD protein of the COVID-19 stealth omicron mutant virus was -12.09 kcal/mol, the inhibition constant was 1.38 nM, and PGG and Corona The average binding energy of the S RBD protein of the -19 stealth omicron mutant virus was -2.45 kcal/mol and the inhibition constant was 15.96 mM.

In addition, as shown in Figure 6, it can be confirmed that the hydrogen bonding sites between the S RBD of COVID-19 Stealth Omicron and geranin or PGG are THR430 and PHE515 for geranin, and ASN360, ASN388, and ASP389 for PGG. there was.

Example III. Inhibitory effect of geranin and PGG on COVID-19 omicron mutant virus S RBD binding

1. Experimental method

Referring to the molecular docking analysis results of Example II, the inhibitory effect of geranin and PGG on RBD-ACE2 binding against the COVID-19 virus and its mutant forms (alpha mutation, beta mutation, delta mutation, and omicron mutation) was evaluated. It was analyzed using the SARS-CoV-2 surrogate virus neutralization test kit according to the manufacturer's protocol.

1-1. Kit contents

Component Quantity Park No. Capture Plate 1 plate S1-80 Negative control (NC) 1 vial (0.05 mL) S1-11 HRP conjugated RBD 1 vial (0.02 mL) S1-30 HRP Dilution Buffer 1 bottle (10 mL) S1-90 Sample Dilution Buffer 1 bottle (30 mL) S1-60 20X Wash Solution 1 bottle (40 mL) S1-70 TMB Solution 1 bottle (12 mL) S1-40 Stop Solution 1 bottle (6 mL) S1-50 Plate Sealer 2 pieces N/A

1-2. Reagent preparation

1) All components of the kit were equilibrated at room temperature (20-25 ℃) before performing the analysis.

2) HRP-RBD preparation: HRP conjugated RBD was diluted at a ratio of 1:999 using HRP Dilution Buffer (Ex, mix 10 μL HRP conjugated RBD and 9990 μL HRP Dilution Buffer to make 10 mL of HRP-RBD solution. ).

3) 1X Wash Solution preparation: 20X Wash Solution was diluted with distilled water at a ratio of 1:19 (Ex, 40 mL 20X Wash Solution and 760 mL distilled water were mixed to make 800 mL of 1X Wash Solution).

4) Negative controls were diluted at a ratio of 1:9 using Sample Dilution Buffer (Ex, made by mixing 10 μL Negative controls and Sample Dilution Buffer), and 1 mg/mL and 10 mg/mL samples (geranin and PGG) were diluted to concentrations of 12.5, 25, and 50 μg/mL using Sample Dilution Buffer.

1-3. Experimental Procedure

1) The diluted sample and negative control in a 24-well plate were mixed with HRP-RBD solution at a ratio of 1:1 and incubated at 37°C for 30 minutes.

2) Add 100 μL of the mixture from 1) to each capture plate, cover the plate with a plate sealer, and incubate at 37°C for 15 minutes.

3) The plate sealer was removed and washed 4 times with 1X wash solution (260 μL).

4) Add 100 μL of TMB solution to each well, block light at room temperature (20-25°C), and react for 15 minutes.

5) 50 μL of Stop solution was added to each well and the absorbance was measured at 450 nm.

1-4. Quality Management

The optical density (OD450) of the negative control must be in the > 1.0 range. If the OD450 value of the control group was not within the range, the test was invalidated and the experiment was repeated.

The percent inhibition of RBD binding was calculated as follows:

RBD binding inhibition rate (%) = (1-OD value of Sample/OD value of Negative control)*100

2. Results

As shown in Figure 7, it was confirmed that both geranin and PGG, the active ingredients of the dampalsu extract, exhibited an excellent RBD-ACE2 binding inhibitory effect between host cells and the COVID-19 omicron mutant virus.

Example IV. Antiviral effect of geranine against COVID-19 omicron mutant virus

To confirm the antiviral effect of geranin against the COVID-19 omicron mutant virus, a dose response curve (DRC) experiment was performed in a COVID-19 omicron mutant virus cell infection model. Infected cells were imaged through immunofluorescence using an antibody specific for the viral nucleocapsid (N) protein, and the obtained images were analyzed using Columbus software (Perkin Elmer).

1. Experimental method

1-1. Viruses and Cell Lines

COVID-19 Omicron mutant strain (NCCP no. 43408, hCoV 19/Korea/KDCA447321/2021) was provided by the Korea Disease Control and Prevention Agency (KDCA), and Vero cells were obtained from ATCC (ATCC-CCL81).

1-2. reagent

Chloroquine, lopinavir, and remdesivir, used as reference compounds, were purchased from Sigma Aldrich, SelleckChem, and MedChemExpress, respectively. The primary antibody specific for anti-SARS-CoV-2 N protein was purchased from Sino Biological, and the secondary antibodies, Alexa Fluor 488 goat anti-rabbit IgG and Hoechst 33342, were purchased from Molecular Probes.

1-3. Dose response curve analysis by immunofluorescence

1.2 × 10 ⁴ Vero cells per well were inoculated into a 384-tissue culture plate. The next day, the cells were treated with compounds prepared at 10 points by serially diluting 2-fold in DMSO so that the maximum concentration was 50 μM. Approximately 1 hour after compound treatment, cells were infected with the COVID-19 Omicron mutant strain at an MOI of 0.045 in a BSL3 facility and cultured at 37°C for 48 hours. Afterwards, cells were fixed with 4% paraformaldehyde (PFA) and permeabilized. Afterwards, cells were stained with anti-SARS-CoV-2 nucleocapsid (N) primary antibody, Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody, and Hoechst 33342. Fluorescent images of infected cells were acquired using Operetta (Perkin Elmer), a high-capacity image analysis device.

1-4. image analysis

The acquired images were analyzed using Columbus software. The total number of cells per well was calculated as the number of nuclei stained with Hoechst, and the number of infected cells was calculated as the number of cells expressing the viral N protein. The infection ratio was calculated as 'number of cells expressing N protein/total number of cells' in each well. The % inhibition of infection of each well is calculated as the average infection rate of wells containing non-infected cells (mock) and the average infection rate of wells containing infected cells treated with 0.5% DMSO (v/v) in the same plate, respectively. It was calculated by normalizing to % and 0%. The cell viability of the compound was expressed in the graph as '% Cell viability' by normalizing the number of cells in each well to the average number of cells in the mock group wells. Each value was obtained through two repeated experiments (technical duplicate). The reaction curve and IC ₅₀ and CC ₅₀ values according to compound concentration were derived using the formula Y = Bottom + (Top - Bottom)/(1 + (IC ₅₀ /X)Hillslope) in XLFit 4 (IDBS) software. Selectivity index (SI) value was calculated as CC ₅₀ /IC ₅₀ .

2. Results

As shown in Figure 8, it was confirmed that geranin, the active ingredient of dampalsu extract, exhibits excellent anti-infective effect (antiviral effect) against the COVID-19 omicron mutant virus. In particular, it showed an effective effect at a much lower concentration compared to remdesivir, chloroquine, and lonafavir, which were selected as comparative substances.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.

Claims (14)

A pharmaceutical composition for preventing or treating COVID-19 omicron mutant virus infection, comprising a compound of the following formula 1 or 2, or a pharmaceutically acceptable salt thereof, as an active ingredient.
[Formula 1]

[Formula 2]

According to paragraph 1,
A pharmaceutical composition, characterized in that the compound is isolated from dampalsu extract.
According to paragraph 2,
A pharmaceutical composition, characterized in that the dampalsu extract is a dampalsu leaf extract.
According to paragraph 2,
The dampal water extract contains water, alcohol with 1 to 6 carbon atoms, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, A pharmaceutical composition, characterized in that it is an extract using at least one solvent selected from the group consisting of hexane, cyclohexane, petroleum ether, subcritical fluid, and supercritical fluid.
According to paragraph 1,
A pharmaceutical composition, wherein the compound binds to the S RBD (spike receptor-binding domain) protein of the COVID-19 omicron mutant virus.
According to clause 5,
A pharmaceutical composition, wherein the S RBD (spike receptor-binding domain) protein is represented by the amino acid sequence of SEQ ID NO: 1.
A food composition for preventing or improving COVID-19 omicron mutant virus infection, comprising a compound of the following formula 1 or 2, or a foodologically acceptable salt thereof, as an active ingredient.
[Formula 1]

[Formula 2]

In clause 7,
A food composition, characterized in that the food composition is a health functional food composition.
In clause 7,
A food composition, characterized in that the compound is isolated from dampalsu extract.
According to clause 9,
A food composition, characterized in that the dampalsu extract is a dampalsu leaf extract.
According to clause 9,
The dampal water extract contains water, alcohol with 1 to 6 carbon atoms, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, A food composition, characterized in that it is an extract using at least one solvent selected from the group consisting of hexane, cyclohexane, petroleum ether, subcritical fluid, and supercritical fluid.
In clause 7,
A food composition characterized in that the compound binds to the S RBD (spike receptor-binding domain) protein of the COVID-19 omicron mutant virus.
According to clause 12,
A food composition, wherein the S RBD (spike receptor-binding domain) protein is represented by the amino acid sequence of SEQ ID NO: 1.
A quasi-drug composition for preventing or improving COVID-19 omicron mutant virus infection, comprising a compound of the following formula 1 or 2, or a pharmaceutically acceptable salt thereof, as an active ingredient.
[Formula 1]


[Formula 2]