KR20230132383A - Pharmaceutical composition for the prevention and treatment of Envelopled viruses comprising Codonopsis, Aster and/or Polygala extracts - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention and treatment of COVID-19 containing plant extracts from the genus Codonopsis , Aster , and/or Polygala as active ingredients, and more specifically, to a pharmaceutical composition for the prevention and treatment of COVID-19. ( Codonopsis ), Aster ( Aster ), and/or Polygala ( Polygala ) A pharmaceutical composition for the prevention and treatment of COVID-19 by a 5-cyclic triterpenoid saponin component of a specific structure contained in plants, and a pharmaceutical composition using the pharmaceutical composition. It is about prevention and treatment methods for COVID-19.

Description

Pharmaceutical composition for the prevention and treatment of enveloped viruses comprising Codonopsis, Aster and/or Polygala extracts}

The present invention relates to a pharmaceutical composition for the prevention and treatment of enveloped virus infections containing plant extracts of the genus Codonopsis , Aster , and/or Polygala as active ingredients. In detail, inhibition of cellular infection and intracellular activity against coronavirus 19 by pentacyclic triterpenoid saponin components of a specific structure contained in Codonopsis , Aster , and/or Polygala plants. It relates to a pharmaceutical composition for preventing and treating coronavirus disease-19 (COVID-19) by inhibiting the production of infectious viruses, and a method for preventing and treating COVID-19 using the pharmaceutical composition.

Corona Virus is a type of enveloped RNA virus. It is a virus whose genetic information consists of ribonucleic acid (RNA) and causes infection in the respiratory and digestive systems of humans and animals. Coronavirus can be easily infected mainly through mucosal transmission and droplet transmission, and generally causes mild respiratory infections in humans, but in rare cases, it can cause fatal infections and even lead to death.

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), a type of the above-mentioned coronavirus, emerged in Wuhan, China and spread rapidly around the world, and the World Health Organization (WHO) classifies diseases infected with the virus as COVID-2. It was named 19. According to recent reports, common symptoms of infection include fever (98.6%), fatigue (69.6%), dry cough (59.4%), lymphopenia (70.3%), prolonged prothrombin time (58%), and increased lactate dehydrogenase. (39.9%), etc. (Wang, D. et al., JAMA, 2000).

It has been reported that SARS-CoV-2 is mainly transmitted through person-to-person contact through respiratory droplets formed by coughing and sneezing, and through the surface of an object contaminated by coughing or sneezing (CDC, C.f.d.c.a.p., How COVID-2020) 19 Spreads, Coronavirus Disease 2019 (COVID-19), 2020), and even asymptomatic infection has been reported to be possible (Yu, P., et al., J Infect Dis, 2020; Hoehl, S., et al., N Engl J Med, 2020; Bendix A., Science Alert, 2020).

As SARS-CoV-2 cases surged around the world, WHO declared a 'Public Health Emergency of International Concern' (PHEIC) on January 30, 2020. As confirmed coronavirus infections continued to increase around the world, WHO declared a 'Public Health Emergency of International Concern' (PHEIC) in March. On the 11th, COVID-19 was declared a pandemic (global pandemic) for the third time in history, following the Hong Kong flu (1968) and the swine flu (2009).

Currently, pharmaceutical companies in several countries, including the United States and the United Kingdom, have been developing vaccines and treatments to prevent or treat SARS-CoV-2, some of which have been approved in some countries and are being administered to the general public. However, in the case of the above vaccines, many problems are emerging due to the use of vaccines that have not gone through complete clinical procedures, such as causing severe side effects or, in extreme cases, the death of the person receiving the vaccine, and due to the emergence and spread of mutant viruses. There is controversy over the effectiveness of vaccines.

Therefore, the clinical effect is uniformly excellent regardless of the patient's characteristics, immunity, condition, etc., and the treatment effect is especially excellent for patients suffering from underlying diseases or the elderly with weak immunity, including young children and the elderly, COVID-19. There is an urgent need to develop a treatment that is specific for 19 and variant viruses and has clinically proven therapeutic effects.

Giulio N. et al., Eur J. Pharmacol. 882:173328 (2020.6.27.)

As a result of the present inventors' diligent efforts to solve the above problems, the five-cyclic triterpenoid saponin component of a specific structure contained in plant extracts of the genus Codonopsis , Aster , and/or Polygala . In addition to strengthening organ immunity and various anti-inflammatory effects related to the bronchial tubes, known previously in relation to this respiratory disease, there are two main routes of entry into coronavirus cells: i) cathepsins in lysosomes, and ii) TMPRSS2 (transmembrane protease serine) in the cell membrane. The present invention was completed by confirming that it blocks coronavirus cell infection by showing inhibition of membrane fusion between coronavirus and cells following cleavage of the coronavirus spike protein by subtype 2).

In other words, the object of the present invention is a plant extract of Codonopsis , Aster , and/or Polygala that inhibits cell entry or cell infection of coronavirus, or 5 of a specific structure contained in the extract. The object of the present invention is to provide a pharmaceutical composition for preventing or treating enveloped viral infections containing cyclic triterpenoid saponin as an active ingredient, and a method for preventing or treating enveloped viral infections using the same.

The present invention provides a pharmaceutical composition for preventing or treating enveloped virus infections, comprising a compound represented by the following formula (1) as an active ingredient.

[Formula 1]

In Formula 1,

R ¹ is hydrogen or hydroxy;

R ² is methyl or hydroxymethyl;

또는

이고;R ³ is

or

ego;

이고;R ^a is hydrogen or

ego;

이고;R ^b is hydrogen or

ego;

또는

이다. ^Rc is

or

am.

In addition, the present invention provides a pharmaceutical composition for preventing or treating enveloped virus infections comprising Codonopsis plant extract, Aster plant extract, Polygala plant extract, or a mixture thereof. provides.

Additionally, the present invention provides a pharmaceutical preparation comprising a pharmaceutical composition for preventing or treating the enveloped virus infection.

The present invention relates to extracts of Codonopsis , Aster , and/or Polygala , and/or pentacyclic triterpenoid saponin components of a specific structure contained in the extracts that prevent coronavirus from entering cells and It was confirmed that it had a significant inhibitory effect on cell infection. In addition, the plant extracts of the Codonopsis , Aster , and/or Polygala genus, and/or the 5-cyclic triterpenoid saponin component of a specific structure contained in the extracts are used to inhibit the interaction between coronaviruses and cells. By effectively inhibiting membrane fusion and blocking cell infection by coronavirus, it has the effect of suppressing the proliferation and spread of infectious coronavirus within cells. In addition, Codonopsis , Aster , and/or Polygala plant extracts, and/or pentacyclic triterpenoid saponin components of a specific structure contained in the extracts help fuse the host cell membrane and the virus membrane. It can inhibit infection by SARS-CoV-2 variants and the parental strain regardless of S mutation status, suggesting potential broad-spectrum anti-SARS-CoV-2 activity. It can be a broad-spectrum antiviral agent that is active even against variants. Therefore, the composition of the present invention is useful for preventing and treating coronavirus.

Figure 1 - Analysis results of (A) inhibition effect of SARS-CoV-2 entry into cells and (B) cell viability analysis results of Codonopsis lanceolata extract
Figure 2 - Structures of eight major compounds isolated from extracts of Deodeok
Figure 3 - Results of analysis of pSARS-CoV-2 entry inhibition effect of eight major compounds isolated from extracts of Deodeok (A); Analysis results of pSARS-CoV-2 entry inhibition effect according to lancemarside A (LA) concentration in ACE2+ and ACE2/TMPRSS2+ H1299 cells (B)
Figure 4 - (A) Infectious activity of WT and D614G mutant SARS-CoV-2. Comparison of infectious activity between WT and D614G mutants of pSARS-CoV-2 virus in ACE2+ and ACE2/TMPRSS2+ H1299 cells; (B) Effect of lancemaside A (LA) on infection of WT and D614G mutants of pSARS-CoV-2 virus in ACE2+ and ACE2/TMPRSS2+ H1299 cells.
Figure 5 - Diagram showing antiviral activity against infection of early SARS-CoV-2 isolate (lineage A) in Vero cells, (top) parental (lineage A) in Vero cells Dose-response analysis of lancemicide A (LA) against parental) SARS-CoV-2. Blue squares represent cell viability; Confocal images of SARS-CoV-2 N protein (green) and cell nucleus (red) at different concentrations of lancemaside A (LA) in Vero cells (bottom)
Figure 6 - A diagram showing the antiviral activity against infection by SARS-CoV-2 variants in Vero cells. (Left) Alpha (B.1.1.7), beta (B.1.351), and delta (B. Dose-response analysis of lancemicide A (LA) against 1.617.2), Omicron (B.1.1.529) SARS-CoV-2 variants. (Right) Confocal images of SARS-CoV-2 N protein (green) and cell nucleus (red) at different concentrations of lancemaside A (LA) in Vero cells.
Figure 7 - (A) Schematic of the monitoring system visualizing SARS-CoV-2 membrane fusion with host cells; (B) Results confirming the effect of lancemaside A (LA) on fusion events; (C) Results of quantitative analysis through flow cytometry
Figure 8 - (A) Schematic diagram of Split-GFP analysis. Ectopic expression of SARS-CoV-2 spike protein by culturing a mixture of ACE2/TMPRSS2+ H1299 cells expressing GFP1-10 or GFP11 induced cell-cell fusion, producing GFP fluorescence; (B) Experimental procedure; (C) Split-GFP analysis results. GFP and blue fluorescence indicate S-mediated intercellular fusion and DAPI-stained nuclei, respectively; (D) Decreased syncytium formation in a concentration-dependent manner upon LA treatment; (E) Confirmation of the inhibitory effect of LA on apoptotic cell death induced by syncytium formation through inhibition of cleavage of caspase-3 and -9 following LA treatment.
Figure 9 - pSARS-CoV-2 entry into 70% EtOH extracts of Aster pseudoglehnii , Aster scaber , Polygala tenuifolia and Aster koraiensis in ACE2+ and ACE2/TMPRSS2+ H1299 cells, respectively. Inhibition assay results.
Figure 10 - pSARS-CoV-2 entry inhibition and cell viability analysis results for 70% EtOH extract of A. koraiensis in ACE2+ and ACE2/TMPRSS2+ H1299 cells.
Figure 11 - pSARS-CoV-2 against Astersponin I (A), a triterpenoid saponin isolated from 70% EtOH extract of A. koraiensis in ACE2+ and ACE2/TMPRSS2+ H1299 cells Entry inhibition and cell viability assay (B).
Figure 12 - Effect of astersaponin I on S-mediated cell-cell fusion using Split-GFP assay. (A) Representative images of GFP-positive cell-cell fusion. Blue indicates DAPI-stained nuclei; (B) Astersaponin I treatment effectively reduces syncytium formation in a concentration-dependent manner (IC ₅₀ 3.9 μM).
Figure 13 - Effect of astesaponin I on infection of WT and D614G mutants of pSARS-CoV-2 in ACE2+ and ACE2/TMPRSS2+ H1299 cells.
Figure 14 - Diagram showing antiviral activity against infection of early SARS-CoV-2 isolates (Ancestral SARS-CoV-2) and SARS-CoV-2 variants in Vero cells. (A) Early SARS-CoV in Vero cells. Dose-response analysis of astesaponin I against -2 isolates (Ancestral SARS-CoV-2) and SARS-CoV-2 variants. Blue diamonds represent cell viability. (B) Confocal images of SARS-CoV-2 N protein (green) and cell nucleus (red) at various concentrations in Vero cells.

The present invention will be described in detail below with reference to the attached tables and drawings.

When drawings are described, they are provided as examples so that the idea of the present invention can be sufficiently conveyed to those skilled in the art. Accordingly, the present invention is not limited to the presented drawings and may be embodied in other forms, and the drawings may be exaggerated to clarify the spirit of the present invention.

At this time, if there is no other definition in the technical and scientific terms used, they have the meaning commonly understood by those skilled in the art to which this invention pertains, and the gist of the present invention is summarized in the following description and accompanying drawings. Descriptions of known functions and configurations that may be unnecessarily obscure are omitted.

Additionally, as used herein, the singular forms “a,” “an,” and “the” are intended to also include the plural forms, unless the context clearly dictates otherwise.

Additionally, units used in this specification without special mention are based on weight, and as an example, units of % or ratio mean weight % or weight ratio.

In addition, in this specification, the expression "comprises" is an open description that has the same meaning as expressions such as "comprises," "contains," "has," or "characterized by" and is not additionally listed. No element, material or process is excluded. Additionally, the expression “consisting substantially of…” means that other elements, materials or processes not listed together with the specified element, material or process may impermissibly affect at least one basic and novel technical idea of the invention. This means that it can be present in an amount that does not have a significant effect. Additionally, the expression “consisting of” means that only the elements, materials, or processes described are present.

As used herein, the terms “ingredient”, “composition”, “composition of compounds”, “compound”, “drug”, “pharmaceutically active agent”, “active agent”, “treatment”, “therapy”, “treatment” or “ “Medicine” is used interchangeably to mean a compound or composition of compound(s) or substances that induces a desired pharmaceutical and/or physiological effect by local and/or systemic action when administered to a subject (human or animal). It is used as.

As used herein, the term “treatment” or “therapy” (as well as different forms thereof) includes prophylactic (eg, prophylactic treatment), curative, or palliative treatment. As used herein, the term “treating” includes alleviating or reducing at least one detrimental or negative effect or symptom of a condition, disease or disorder. The terms “prevention,” “improvement,” and “treatment” of the present invention should be interpreted in the broadest sense, and “prevention” refers to patients who may be exposed to or susceptible to a disease but have not yet experienced or revealed symptoms of the disease. This means preventing one or more of the clinical symptoms of the disease from progressing. “Treatment” means any action that arrests or reduces the development of a disease or one or more clinical symptoms thereof.

In this specification, “sample” or “sample” refers to an object for analysis and is used with the same meaning throughout the specification.

Hereinafter, the pharmaceutical composition for preventing or treating enveloped virus infections according to the present invention will be described in detail.

The pharmaceutical composition for preventing or treating envelope-type viral infections according to the present invention contains plant extracts of Codonopsis , Aster , and/or Polygala or mixtures thereof as active ingredients.

Codonopsis plants are perennial herbaceous plants belonging to the Campanulaceae family. About 40 to 50 species are widely distributed throughout Asia, from Pakistan and the Himalayas to China and Japan. Plants of the Deodeok genus that grow naturally in Korea include Codonopsis lanceolata , Codonopsis lanceolata for emaculata , Codonopsis pilosula, Codonopsis minima , and Codonopsis ussuriensis . In particular, Codonopsis lanceolata (CL) has been widely used in the private sector for food and medical purposes since its efficacy and safety were verified a long time ago in East Asian countries such as Korea, China, and Japan. Codonopsis lanceolata (CL) has shown therapeutic activity against various diseases, including bronchitis, cough, and lung damage, but its antiviral activity, especially its antiviral effect specific to coronaviruses, has not been revealed.

Aster plants are perennial herbaceous plants belonging to the Asteraceae family. Plants in the genus Aster include Aster pekinensis , Aster hayatae , Aster tripolium , and Aster fastigiatus . , Danyang Aster ( Aster altaicus var. uchiyamae ), Aster associatus , Jeju chrysanthemum ( Aster chezuensis ), Aster scaber , Aster sphathulifolius , Aster koraiensis, Aster. pseudoglehnii ), Aster tataricus , Aster maackii , Aster ageratoides, Aster hispidus , Aster incisus , Aster arenarius, Aster aster ( Aster arenarius ) There are Aster lautureanus , Aster magnus , Aster meyendorii, Aster novibelgii , and Aster yomena . Aster scaber is rich in calcium, iron, and vitamin A, and has been used as an herbal ingredient since ancient times. It has also been used as a herbal medicine to treat pain relief, dizziness, detoxification, back pain, and enteritis. Aster koraiensis is a perennial plant of the Aster family. It is also called Korean mugwort. It is a Korean endemic species and is also called Korean Daisy. Young shoots are eaten as a vegetable, and the whole plant is known to have antibacterial properties. However, nothing has been revealed about its antiviral activity, especially its antiviral effect specific to coronaviruses.

Polygala plants are herbs belonging to the Polygalaceae family, and include the annual grasses Polygala tatarinowii and the perennial grasses Polygala japonica , Polygala sibirica , and Polygala tenuifolia . . Polygala tenuifolia is known to have expectorant, sedative, blood pressure lowering, and hemolytic effects.

The present invention discloses the effect of extracts from Codonopsis , Aster , and/or Polygala plants to inhibit cell entry of coronaviruses, and applies them for the prevention or treatment of coronaviruses. There is a characteristic. In particular, in one embodiment of the present invention, while plant extracts of Codonopsis , Aster , and/or Polygala do not inhibit the entry of another virus, vesicular stomatitis virus (VSV), coronavirus It was confirmed that it has a coronavirus-specific cell entry inhibitory effect by inhibiting the interaction of the spike protein and its receptor.

The term "extract" in the present invention refers to a substance obtained by separating from Codonopsis , Aster , and/or Polygala plants.

In one embodiment, the plants of the Codonopsis genus include Codonopsis lanceolata , Codonopsis lanceolata for emaculata , Codonopsis pilosula, Codonopsis minima , and Codonopsis . ussuriensis ).

In one embodiment, the Aster plants include Aster pekinensis , Aster hayatae , Aster tripolium , Aster fastigiatus , and Aster altaicus var. uchiyamae ), Aster associatus , Jeju chrysanthemum ( Aster chezuensis ), Aster scaber , Aster sphathulifolius , Aster koraiensis , Aster pseudoglehnii , Aster tataricus , Aster maackii , Aster ageratoides , Aster hispidus, Aster incisus , Aster arenarius , Aster lautureanus , Aster lautureanus. It may be any one or more selected from Aster magnus , Aster meyendorfii , Aster novibelgii , and Aster yomena .

In one embodiment, the Polygala plant may be any one or more selected from Polygala tatarinowii , Polygala japonica, Polygala sibirica , and Polygala tenuifolia .

The Codonopsis , Aster , and/or Polygala plants can be purchased commercially, or those grown or collected from the natural world can be used. Additionally, the parts of each plant used are not limited, and all parts of each plant, including leaves, stems, roots, flowers, and fruits, can be used.

In one embodiment, the Codonopsis , Aster , and/or Polygala plant extract is derived from Codonopsis , Aster , and/or Polygala plants. It is obtained by extraction with a solvent selected from the group consisting of water, C1 to C4 alcohol, and mixed solvents of water and C1 to C4 alcohol, preferably Codonopsis , Aster , and /Or, the Polygala plant extract may be obtained by extraction with a mixed solvent of water and C1-C4 alcohol, preferably methanol or ethanol.

The mixed solvent is preferably about 70% ethanol aqueous solution. Extraction may be performed by extraction methods known in the art, such as cold immersion, hot water extraction, ultrasonic extraction, reflux cooling extraction, etc., but is not limited thereto. As for the extraction temperature, a person skilled in the art may adopt various temperature ranges appropriate for the extraction method, for example, it may be performed at 20°C to 100°C, but is not limited thereto. In addition, the extraction time varies depending on the extraction method, and a person skilled in the art can select an appropriate extraction time, but is not limited to this, and can be performed once or multiple times in the range of about 1 hour to 10 days. Preferably, the extraction can be performed by extracting with the above-mentioned extraction solvent 2 to 3 times for about 2 days at room temperature.

There is no limitation to the method for producing the extract of the present invention, and any known method can be used. For example, the extract included in the composition of the present invention can be prepared in powder form from the primary extract extracted by the above-described hot water extraction or solvent extraction method by additional processes such as reduced pressure distillation and freeze-drying or spray drying. In addition, the primary extract was further purified into fractions using various chromatographies such as silica gel column chromatography, thin layer chromatography, and high performance liquid chromatography. You can also get it. Therefore, in the present invention, extract is a concept that includes all extracts, fractions, and purifications obtained at each stage of extraction, fractionation, or purification, as well as their dilutions, concentrates, or dried products.

In the present invention, the plant extract of Codonopsis , Aster and/or Polygala is an extract, a diluted or concentrated solution thereof, a dry powder of the extract, or any form formulated using the same. It may be included, and preferably may be in a formulation such as granules, powder, or pellets.

In one embodiment, the Codonopsis extract may be an extract of Codonopsis lanceolata , which may be an extract extracted from the roots and/or root bark of Codonopsis lanceolata , but is not limited thereto. , it may be extracted from leaves, stems, etc.

In one embodiment, the Aster extract may be an extract of Aster scaber , Aster koraiensis , or Aster pseudoglehnii , which is an extract of Aster scaber , Aster koraiensis . Alternatively, it may be an extract extracted from the leaves of Aster pseudoglehnii , but is not limited to this and may be extracted from the roots, stems, flowers, etc.

In one embodiment, the Polygala extract may be an extract of Polygala tenuifolia , which may be an extract extracted from the roots of Polygala tenuifolia , but is not limited thereto, and includes leaves, stems, It may be extracted from flowers, etc.

In the present invention, the composition may contain additional ingredients necessary for administration, effect, etc., in addition to plant extracts of Codonopsis , Aster , and/or Polygala , and may contain other coronavirus infections. It can be used in combination with inhibitors, treatments, vaccines, neutralizing antibodies, etc.

The plant extract of Codonopsis , Aster , and/or Polygala of the present invention contains a compound represented by the following formula (1).

[Formula 1]

In Formula 1,

R ¹ is hydrogen or hydroxy;

R ² is methyl or hydroxymethyl;

또는

이고;R ³ is

or

ego;

이고;R ^a is hydrogen or

ego;

이고;R ^b is hydrogen or

ego;

또는

이다. ^Rc is

or

am.

The pharmaceutical composition for preventing or treating envelope-type viral infections according to the present invention contains the compound represented by Formula 1 above as an active ingredient.

In one embodiment, the compound of Formula 1 may be represented by Formula 2 below.

[Formula 2]

이다.In Formula 2, R ^a is hydrogen or

am.

In one embodiment, the compound of Formula 1 may be represented by Formula 3 below.

[Formula 3]

In one embodiment, the Codonopsis plant extract includes a pentacyclic triterpenoid saponin compound represented by Chemical Formula 2.

In an embodiment, the Codonopsis plant extract includes compounds of Formula 2A and Formula 2B below.

[Formula 2A]

[Formula 2B]

The compound of Formula 2A is named lancemaside A (LA), and the compound of Formula 2B is named lancemaside B.

In an embodiment, the Codonopsis plant extract may be a Codonopsis lanceolata extract.

In one embodiment, the Aster plant extract includes a pentacyclic triterpenoid saponin compound represented by Chemical Formula 3.

The compound of Formula 3 is named astesaponin I.

In an embodiment, the Aster plant extract may be an Aster scaber extract, an Aster koraiensis extract, or an Aster pseudoglehnii extract, preferably an Aster koraiensis extract. there is.

In an embodiment, the envelope-type virus includes Severe Acute Respiratory Syndrome-Corona Virus-2 (SARS-CoV-2), Middle East Respiratory Syndrome (MERS) virus, and Severe Acute Respiratory Syndrome (Severe Acute Respiratory Syndrome, SARS) virus, and more specifically, severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2).

The term “salt” herein refers to an addition salt of an inorganic acid salt, organic acid salt, or metal salt of a compound, and when used in a pharmaceutical composition, it may be a “pharmaceutically acceptable salt.” The pharmaceutically acceptable salt may be a salt that does not cause serious irritation to the organism to which the compound is administered and does not impair the biological activity and physical properties of the compound. As used herein, the term “pharmaceutically acceptable salt” refers to a formulation of a compound that does not cause significant irritation to the organism to which the compound is administered and does not impair the biological activity and physical properties of the compound. The pharmaceutically acceptable salts include acids that form non-toxic acid addition salts containing pharmaceutically acceptable anions, for example, inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, tartaric acid, and formic acid. , organic carbonic acids such as citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicic acid, etc., methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. Acid addition salts formed with the same sulfonic acid, etc. are included. For example, pharmaceutically acceptable carboxylic acid salts include metal or alkaline earth metal salts formed by lithium, sodium, potassium, calcium, magnesium, etc., amino acid salts such as lysine, arginine, guanidine, dicyclohexylamine, N Organic salts such as -methyl-D-glucamine, tris(hydroxymethyl) methylamine, diethanolamine, choline, and triethylamine are included. Although acids such as oxalic acid are not pharmaceutically acceptable, they can be used as intermediates for obtaining the compounds of the present invention and their pharmaceutically acceptable salts, and in the preparation of useful salts.

The plant extract of Codonopsis , Aster , and/or Polygala of the present invention, and/or the pentacyclic triterpenoid saponin component of a specific structure contained in the extract is the spike protein of the coronavirus. It can be characterized by inhibiting i) the endocytosis pathway by ACE2 (angiotensin converting enzyme 2) and ii) the direct fusion pathway by TMPRSS2 and ACE2, which are related to cell entry through .

The plant extract of Codonopsis , Aster and/or Polygala of the present invention, and/or the 5-cyclic triterpenoid saponin component of a specific structure contained in the extract is the TMPRSS2 and/or It may be characterized by inhibiting or reducing the interaction of ACE2 with the coronavirus, especially the spike protein of the coronavirus.

In one embodiment of the present invention, the coronavirus used was the SARS-CoV-2 virus, which is the virus that causes COVID-19, but was recombined to express a spike protein similar to the spike protein of the SARS-CoV-2 virus. It was also confirmed that it inhibits virus entry into cells. Therefore, the plant extract of Codonopsis , Aster , and/or Polygala of the present invention, and/or the 5-cyclic triterpenoid saponin component of the specific structure contained in the extract (Formula 2A, 2B, 3) is not only SARS-CoV-2, but also other coronaviruses or viruses of other genera that express spike proteins similar to SARS-CoV-2 and enter cells through interaction with ACE2 and/or TMPRSS2. It may be characterized as having the ability to suppress and prevent or treat the viral infection. For example, it can effectively inhibit cell entry of MERS virus, SARS-CoV1 virus, etc.

In the present invention, the “cell” refers to a cell that can be infected with a virus, especially a coronavirus. The cells may be plant or animal cells, and may be isolated cells or cells that are not isolated from living organisms, organs, or tissues. Preferably they are mammalian cells, and more preferably human cells. In the present invention, the cells are preferably respiratory cells, but are not limited thereto.

According to one embodiment of the present invention, the cells may include human lung epithelial cells (H1299, A549, Calu-3, etc.), such as human lung fibroblasts (MRC-5), human colon (CaCo2), It may include, but is not limited to, human kidney (HEK293T), monkey kidney (Vero) cells, etc., and cells that can achieve a SARS-CoV-2 entry blocking effect by administering the active ingredient or composition according to the present invention If so, it can be said to be included in the above category.

The pharmaceutical composition for preventing or treating envelope-type viral infections according to the present invention may be a pharmaceutical composition for preventing or treating severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infections.

In the pharmaceutical composition for preventing or treating severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection according to an example of the present invention, the Codonopsis genus, Aster genus and/or Polygala plant extract, and/or the pentacyclic triterpenoid saponin component with a specific structure contained in the extract inhibits cell entry of SARS-CoV-2 and/or binds to the SARS-CoV-2 spike protein. It may be suppressing.

In the pharmaceutical composition for preventing or treating severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection according to an example of the present invention, the Codonopsis genus, Aster genus and/or Polygala plant extract, and/or the pentacyclic triterpenoid saponin component of a specific structure contained in the extract may be included in an amount of 0.001 to 50% by weight based on the total weight of the composition.

The pharmaceutical composition for preventing or treating severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) according to the present invention includes the compound of Formula 1 above.

In the pharmaceutical composition for preventing or treating severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection according to an example of the present invention, the compound of formula 1 is used to inhibit SARS-CoV-2 from entering cells and /Or it may inhibit binding to the SARS-CoV-2 spike protein.

In the pharmaceutical composition for preventing or treating severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection according to an example of the present invention, the compound of Formula 1 is present in an amount of 0.001 to 50 weight based on the total weight of the composition. %, more preferably 1 to 30% by weight, and even more preferably 3 to 20% by weight, but is not limited thereto, and may be used by adjusting appropriately if the maximum preventive or therapeutic effect can be achieved without causing side effects. Of course it is possible.

The pharmaceutical composition according to the present invention contains the Codonopsis extract, Aster extract, Polygala extract or a mixture thereof, or a conventional pharmaceutical composition in addition to containing the compound of Formula 1. It may further include suitable carriers, excipients and diluents used in the composition.

Carriers, excipients and diluents that may be included in the composition include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, These include methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. When formulating the composition, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

The pharmaceutical composition according to the present invention can be formulated and used in various forms according to conventional methods. Suitable dosage forms include oral dosage forms such as tablets, pills, powders, granules, dragees, hard or soft capsules, solutions, suspensions or emulsions, injections, and aerosols. Among these, granules and powder pellets are preferred. This is most desirable. Other dosage forms include external preparations, suppositories, and sterile injection solutions, but are not limited thereto.

The pharmaceutical composition according to the present invention can be prepared in a suitable dosage form using a pharmaceutically inert organic or inorganic carrier. That is, if the dosage form is a tablet, coated tablet, dragee, or hard capsule, it may contain lactose, sucrose, starch or a derivative thereof, talc, calcium carbonate, gelatin, stearic acid, or a salt thereof. Additionally, when the dosage form is a soft capsule, it may contain vegetable oil, wax, fat, semi-solid and liquid polyol. Additionally, when the formulation is in the form of a solution or syrup, it may include water, polyol, glycerol, and vegetable oil.

In addition to the above carrier, the pharmaceutical composition according to the present invention may further include preservatives, stabilizers, wetting agents, emulsifiers, solubilizers, sweeteners, colorants, osmotic pressure regulators, antioxidants, etc.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount.

As used herein, “pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, and activity of the patient's disease. , 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 field of medicine. 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 maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

The composition of the present invention can be used in combination with various treatments that can be recognized by those skilled in the art as being effective in preventing or treating coronavirus infections and pharmaceutical compositions for treating other coronavirus infections.

The pharmaceutical composition of the present invention can be administered to an individual through various routes. The mode of administration may be, for example, oral administration. It may be administered by subcutaneous, intravenous, intramuscular or intrauterine intrathecal or intracerebrovascular injection. The pharmaceutical composition of the present invention is determined depending on the type of drug that is the active ingredient, along with various related factors such as the disease to be treated, the route of administration, the patient's age, gender and weight, and the severity of the disease.

The present invention provides a pharmaceutical formulation comprising a pharmaceutical composition for preventing or treating the enveloped virus infection.

In the pharmaceutical preparation according to an example of the present invention, the pharmaceutical preparation may be in a solid dosage form or a liquid dosage form.

In the pharmaceutical formulation according to an example of the present invention, the pharmaceutical formulation may be for oral, parenteral, intranasal, oral, buccal, sublingual, airway spray, or rectal administration.

In the pharmaceutical preparation according to an example of the present invention, the pharmaceutical preparation may be in the form of a capsule, pill, tablet, solution, suspension, spray, foam, patch, or paste.

The method of treating enveloped virus infection according to the present invention includes administering the pharmaceutical composition to an individual who has developed or is at risk of developing enveloped virus infection.

The method for treating envelope-type virus infection according to the present invention is to treat individuals who have developed or are at risk of developing envelope-type virus infection, using Codonopsis extract, Aster extract, Polygala extract, or mixtures thereof. It includes the step of administering.

The method for treating enveloped virus infection according to the present invention includes administering the compound of Formula 1 to an individual who has developed or is at risk of developing enveloped virus infection.

As used herein, the term “individual” refers to all animals, including humans, that have developed or are likely to develop enveloped virus infection. The animal may be not only a human, but also a mammal such as a cow, horse, sheep, pig, goat, camel, antelope, dog, or cat that requires treatment for similar symptoms, but is not limited thereto.

As used herein, the term "administration" refers to administering the pharmaceutical composition of the present invention, Codonopsis , Aster , Polygala plant extract or mixtures thereof, or the compound of Formula 1 to an individual by any appropriate method. It means supplying, and the administration route can be administered through various routes such as oral, parenteral, inhalation, sublingual, transdermal, and intranasal administration as long as it can reach the target tissue.

In an embodiment, the envelope-type virus includes Severe Acute Respiratory Syndrome-Corona Virus-2 (SARS-CoV-2), Middle East Respiratory Syndrome (MERS) virus, and Severe Acute Respiratory Syndrome (Severe Acute Respiratory Syndrome, SARS) virus, and more specifically, severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2).

Specifically, the administration may be oral, parenteral, intranasal, oral, buccal, sublingual, airway spray, or rectal.

When the administration is performed by airway spray, the Codonopsis , Aster , and/or Polygala plant extracts, and/or the compound of Formula 1 are applied to the respiratory tract to prevent SARS-CoV- 2 This may indicate an infection prevention effect.

For example, when administered via airway spray, the Codonopsis , Aster , and/or Polygala plant extracts, and/or the compound of Formula 1 are used in an amount of 1 to 20% based on the total weight of the composition. It may be included in weight percent.

The Codonopsis , Aster , and/or Polygala plant extracts may be in the form of granules or powder.

In one embodiment, the Codonopsis , Aster , and/or Polygala plant extract may be in the form of granules or powder for respiratory spraying.

Hereinafter, the contents of the present invention will be described in more detail through examples. The examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited thereto.

[Cell culture]

- H1299 cells were obtained from the Korean Cell Line Bank and cultured in RPMI-1640 (Gibco, USA).

- HEK293T cells were obtained from the American Type Culture Collection (ATCC, USA, CRL-3216). Vero cells were obtained from the American Type Culture Collection (ATCC, USA, CCL-81). HEK293T cells and Vero cells were grown in DMEM medium (Dulbecco's Modified Eagle's Medium; Corning, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1X penicillin-streptomycin solution (HyClone, USA) with 5% CO ₂ It was cultured in a humidified 37°C constant temperature incubator containing .

[Plant Material]

Codonopsis lanceolata Trautv. The above-ground parts and roots of Campanulaceae (Campanulaceae) were collected from Daemosan Mountain in Seoul, South Korea in April 2021. The origin of the plant material was authenticated by one of the authors, DSJ, and the voucher specimens (COLA-A-2021 and COLA-R-2021) were deposited at the College of Pharmacy, Kyunghee University, South Korea.

In May 2021, leaves of Aster koraiensis (voucher specimen #: ASKO2-2021) were collected at Maru Park, Gangnam-gu, Seoul, Korea. Above-ground parts of Aster pseudoglehnii Y.Lim, JOHyun & H.Shin (voucher specimen #: ASPS2-2021) and Aster scabra Thunb. (voucher specimen #: ASSC1-2021) were collected in Seoul in May 2021. Purchased at a grocery store in Dongdaemun-gu. The roots of Polygala tenuifolia Willd. (voucher specimen #: POTE1-2016) were obtained from Hyunjin Pharmaceutical, Dongdaemun-gu, Seoul, Korea, in October 2016. The origin of the plant material was authenticated by one of the authors, DSJ, and the voucher specimen was deposited at the College of Pharmacy, Kyung Hee University, South Korea.

[Extraction and Isolation]

Deodeok ( Codonopsis lanceolata )

Fresh aerial parts (289 g) and root parts (209 g) of Codonopsis lanceolata were sliced and extracted five times in an ultrasonic bath (3 L and 2 L of 70% EtOH, respectively, for 2 h at room temperature). did. The extract was filtered and concentrated using a rotary evaporator to obtain 70% EtOH extract of the aerial part (29.4 g, 10.20%) and 70% EtOH extract of the root (23.5 g, 11.24%), respectively.

Eight compounds were tested: tangshenoside I (1), ethylsyringin (2), syringin (3), sinapaldehyde glucoside (4), lobetyolin (5), lancemaside A (6), lancemaside B (7), and lancemaside D (8). )) were isolated from the roots of CL, and their isolation process and structure confirmation method were as described in previously published references (Chemical constituents of the roots of Codonopsis lanceolata . Du YE, Lee JS, Kim HM, Ahn JH , Jung IH, Ryu JH, Choi JH, Jang DS. Arch Pharm Res. 2018, 41(11):1082-1091.).

Bee ant smell ( Aster koraiensis )

Fresh leaves (320 g) of Aster koraiensis were sliced and extracted three times with 70% EtOH (3 L) in an ultrasonic bath at room temperature for 2 hours. The extract was filtered and evaporated in vacuum at 45°C to obtain a 70% EtOH extract (17.21 g, 5.38%) of Aster koraiensis .

In 2017, leaves of Aster koraiensis were collected for the isolation of triterpenoid saponin in Pyeongchang, Gangwon-do, Korea. Dried and ground Aster koraiensis leaves (5.0 kg) were extracted twice with 95% EtOH (25 L) at 70°C for 3 hours, and the extract solution was removed using a rotary evaporator. The 95% EtOH extract (500 g) was chromatographed on a Diaion HP-20 (ф9.8 × 63.0 cm) eluting with an acetone-H ₂ O gradient (from 0:1 to 1:0 v/v) to yield 28 fractions ( C1 to C28) were obtained. Fraction C11 was separated into seven subfractions by column chromatography using Sephadex LH-20 (ф×65.0 cm), and astersaponin I (96.5 mg) was isolated using 50% acetone. The chemical structure of the isolated material was identified through comparative analysis with ¹ H- and ¹³ C-NMR data and data from references that had already been reported for separation from Aster koraiensis (An Autophagy Inducing Triterpene Saponin Derived from Aster koraiensis . Kwon J, Ko K, Zhang L, Zhao D, Yang HO, Kwon HC. Molecules. 2019, 24(24):4489).

Tuna ( Aster scaber )

Fresh leaves (92 g) of Aster scaber were sliced and extracted three times with 70% EtOH (1.5 L) in an ultrasonic bath at room temperature for 2 hours. The extract was filtered and evaporated in vacuum at 45°C to obtain a 70% EtOH extract (3.70 g, 4.02%) of Aster scaber .

Snail ( Aster pseudoglehnii )

Fresh leaves (150 g) of Aster pseudoglehnii were sliced and extracted three times with 70% EtOH (2 L) in an ultrasonic bath at room temperature for 2 hours. The extract was filtered and evaporated in vacuum at 45°C to obtain a 70% EtOH extract (6.94 g, 4.63%) of Aster pseudoglehnii .

Raw paper ( Polygala tenuifolia )

The dried roots (400g) of polygala tenuifolia were extracted twice for 3 hours at 80°C with 70% EtOH (4L) in a water bath, and the solvent was removed using a rotary evaporator at 45°C to produce polygala tenuifolia . A 70% EtOH extract (84g) was obtained.

[Plasmids]

- pCEP4-myc-ACE2 plasmid was donated by Erik Procko (Addgene plasmid #141185).

- SARS-CoV-2 spike plasmid was donated by Fang Li (Addgene plasmid #145032).

- pHR-CMV and pHR-CMV_IRES-EmGFP lentiviral expression vectors were produced by A. It was a gift from A. Radu Aricescu (Addgene plasmids #113887 and #113888).

Full length of the ACE2 sequence from pCEP4-myc-ACE2, full length of the SARS-CoV-2 S sequence from the SARS-CoV-2 spike plasmid, and the firefly luciferase gene with restrictions of Eco RI and Age l. It was cloned into pHR-CMV or pHR-CMV_IRES-EmGFP lentiviral expression vector using the restriction site.

- TMPRSS2 lentiviral expression vector, RRL.sin.cPPT.SFFV/TMPRSS2 (variant 1).IRES-neo.WPRE (MT130) was a gift from Caroline Goujon (Addgene plasmid # 145843).

[Cell line establishment]

For gene silencing, shRNA sequences containing targeting NPC1 (5'-CCAGGTTCTTGACTTACAA-3'), NPC2 (5'-CGGTTCTGTGATGGAGTTAT-3') and non-specific (NS) (5'-CAACAAGATGAAGAGCACCAA-3') The oligonucleotide was cloned into the pLKO.1 puro lentivirus shRNA plasmid. For stable cell line generation, the plasmid was transiently transfected into HEK293T cells with packaging plasmid psPAX2 and envelope plasmid pMD2.G using Lipofectamine 3000 transfection reagent (Thermo Fisher, USA) according to the manufacturer's instructions. I ordered it.

At 24 and 48 hours after transfection, the virus particle-containing supernatant was harvested, filtered through a 0.45 μm-pore-size filter and used to infect screened H1299 cells on 6-well plates at 50 to 60% confluence. Cultured until confluence was reached. 1 ml viral supernatant was overlaid directly on cells in the presence of polybrene (Merck, Germany) at a final concentration of 4 μg/ml. After 24 hours, the supernatant was replaced with new medium and cultured for 2 to 3 days.

Generation of SARS-CoV-2 pseudovirus and pSARS-CoV-2 entry assay (SARS-CoV-2 pseudovirus and pSARS-CoV-2 entry assay)

SARS-CoV-2 S-pseudotyped lentiviruses (pSARS-CoV-2) were generated using a second generation lentivirus packing system [Exp Mol Med. 53, 956-972 (2021))].

HEK293T cells reaching 70 to 80% confluency in 6-well plates were incubated with pHR-CMV-firefly luciferase plasmid, psPAX2 packing plasmid, using Lipofectamine 3000 reagent (Invitrogen, USA) according to the manufacturer's instructions. and transfected with SARS-CoV-2 spike (SARS-CoV-2 S) plasmid.

At 24 and 48 hours after transfection, viral supernatants were harvested, centrifuged at 500 x g for 5 min to remove detached cells, and stored at 4°C until use.

For pSARS-CoV-2 entry analysis, H1299 cells stably expressing ACE2 (ACE2+) or ACE2 + TMPRSS2 (ACE2/TMPRSS2+) cells were seeded in 48-well plates at 60-80% confluency. The next day, cells were pretreated with the extract or compound for 1 hour, and then the supernatant was removed and replaced with supernatant containing pSARS-CoV-2 virus particles in the presence of the extract or compound. After 24 hours of incubation, pSARS-CoV-2 entry efficiency was determined by measuring firefly luciferase activity in cell lysates using a luciferase assay kit (Promega, USA) and a SpectraMax iD5 multi-mode microplate reader (Molecular Devices, USA). was quantified.

For experiments comparing the infectivity of WT and D614G mutants of SARS-CoV-2, the D614G mutant construct was generated by PCR-based mutagenesis using Pfu DNA polymerase. .

The sense primer sequence for the point mutation is as follows: 5'-GTG GCC GTG CTG TAC CAG GGC GTG AAT TGC ACC GAG GTG-3'.

Culture media containing WT and D614G pSARS-CoV2 viruses were prepared as described above, and ultracentrifugation was performed in a Beckman SW28 rotor at 28,000 rpm for 2 hours at 4°C. The virus pellet was resuspended in PBS buffer, and the virus titer was determined using a qPCR lentivirus titration kit (LV900, ABM). Equivalent viral titers of WT and D614G were used in the pSARS-CoV-2 entry assay.

Virus and dose-response curve (DRC) analysis by immunofluorescence assay

There are three lineages of the SARS-CoV-2 virus, lineage A (hCoV-19/Korea/KCDC03/2020), lineage B.1.1.7 (hCoV-19/Korea/KDCA51463/2021), and lineage B. 1.351. (hCoV-19/Korea/KDCA55905/2021) was provided and used by KDCA. They were propagated in Vero E6 cells, and the virus titer was determined through plaque assay in Vero (TMPRSS2-) cells. All experiments were performed using BSL-3 procedures in a laboratory approved for use by the KDCA in an enhanced biosafety level 3 (BSL-3) isolation facility at the Institut Pasteur Korea.

For dose-dependent curve (DRC) analysis, Vero cells were seeded at 1.2 × 10 ⁴ cells per well in a black 384-well, μClear plate (Greiner Bio-One, Austria). After 24 hours, cells were transferred to a BSL-3 isolation facility, viable SARS-CoV-2 was added at a multiplicity of infection (MOI) of 0.008, and cultured for an additional 24 hours.

The cells were then fixed using 4% paraformaldehyde (PFA), immunostained with an antibody against SARS-CoV-2 nucleocapsid (N) protein, and , cell nuclei were visualized with DNA fluorochrome Hoechst 33342 and analyzed by immunofluorescence analysis.

Images were acquired using an Operetta high-throughput imaging device (Perkin Elmer), and the acquired images were analyzed with Columbus software (PerkinElmer, Inc. Waltham, MA) to quantify cell number and infection rate. Antiviral activity was normalized to the infection control (0.5% DMSO) in each assay plate.

DRCs were generated using Prism7 software (GraphPad). Half maximum inhibitory concentration (IC ₅₀ ) values were determined in duplicate and calculated using nonlinear regression analysis.

Cell viability assay

H1299 and Vero cells (5×10 ³ cells/well) were seeded in a 96-well plate, and treated the next day with plant extracts or the designated concentrations of each compound. After 24 hours of incubation, WST-8 solution (Biomax, Korea) was added to each well and cultured in a humidified 37°C constant temperature incubator containing 5% CO ₂ . The absorbance of each well was measured at 450 nm using a SpectraMax iD5 multi-mode microplate reader (Molecular Devices, USA). Relative cell viability (%) was calculated as the ratio between the average absorbance value of drug-treated samples to the average absorbance value of control cells treated with DMSO.

Cell fusion assay using flow cytometry

ACE2/TMPRSS2+ H1299 cells co-expressing mRuby2 fluorescence were plated in 12 well plates (2 × 10 ⁵ cells/well). After overnight incubation, cells were treated with 10 μM of each compound for 30 min, then 2 × ¹⁰ H1299 cells co-expressing S protein and GFP (Spike-H1299) were added to the culture and incubated for another 1 h. did. The cell mixture was harvested using trypsin-EDTA (Gibco, USA), and 1 × ¹⁰⁴ cells for each condition were analyzed by flow cytometry (GFP/mRuby double-cell by LSRFortessa™) as a measure of cell fusion. The percentage of double-positive cells was determined and data were analyzed using FlowJo software.

Split-GFP fusion assay

Two fragments of GFP, GFP1-10 (residues 1-214) and GFP11 (residues 215-230), were expressed in ACE2/TMPRSS2+ H1299 cells by the lentiviral expression system as described in reference (EMBO J. 40, e106267 (2020)). It was expressed separately in

Equal numbers of ACE2/TMPRSS2+ H1299-GFP1-10 and -GFP11 were inoculated into clear-bottom 96-well plates (ibidi, #89626), infected with lentiviral particles expressing the S protein the next day, and significant numbers of GFP-positive ( The cells were cultured for 48 hours until GFP-positive) cells were formed.

To determine the effect of astesaponin I on the formation of S-mediated syncytium, cell cultures were treated with various concentrations of astesaponin I 6 hours after S protein expression through lentivirus infection. treated with and cultured for an additional 42 hours. Then, cells were fixed with 4% PFA and stained with DAPI (Invitrogen, D1306) to visualize cell nuclei.

After acquiring cell images at ×20 magnification using the ImageXpress Pico automated cell imaging system (Molecular Devices), the cell scoring function of the CellReporterXpress software (nuclear staining and GFP signals were analyzed). Based on this, 2-channel analysis for cell scoring was analyzed. Automated image analysis determined that the population of GFP-positive syncytia reached approximately 30% of the total number of cells plated upon expression of the S protein.

In the field of view, GFP-positive cells appeared as multinucleated syncytia. Nuclei and free nuclei contained in GFP-positive cells were automatically pseudocolored in green and red, respectively, and the percentage of fused cells was calculated as the ratio of green nuclei to red nuclei.

statistical analysis

Data herein are representative of two or three independent experiments with three samples and are expressed as mean ± SEM. Statistical analysis was performed using Student's t-test or one-way ANOVA followed by Tukey's post hoc test.

, p-values less than 0.05 were considered statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant not).

All statistical analyzes were performed using Prism v.9.0.0 (GraphPad Software).

Experimental Example 1: Deodeok ( Codonopsis lanceolata ) Analysis of the inhibitory effect of the extract on SARS-CoV-2 entry into cells

HIV-based lentiviral particles used as a model system for SARS-CoV-2 entry to determine whether extracts of the root part and aerial part of Deodeok inhibit cellular entry of SARS-CoV-2. A pseudovirus expressing the S protein was used (pSARS-CoV-2). The pseudovirus used a model host cell line that stably overexpresses host cell receptor angiotensin-converting enzyme 2 (ACE2) (ACE2+) or ACE2 + TMPRSS2 (ACE2/TMPRSS2+) in H1299 cells. .

ACE2-H1299 cells and ACE2/TMPRSS2-H1299 cells were treated with each extract at various concentrations for 1 hour and then infected with pSARS-CoV-2 pseudovirus. After 24 hours, firefly luciferase activity was measured in cell lysates to quantify the inhibition rate of pSARS-CoV-2 entry.

The 70% EtOH extract of the roots and aerial parts of Deodeok prevented pSARS-CoV-2 from entering ACE2-overexpressing H1299 human lung cancer cells (ACE2+) in a dose-dependent manner, with a half-maximal inhibitory concentration (IC50) of 251 μg/ml, respectively. and 540 μg/ml (Figure 1A). From this, it was confirmed that Deodeok contains a substance with inhibitory activity against SARS-CoV-2 infection, and that this substance is contained in greater amounts in the roots than in the above-ground part. In addition, as shown in Figure 1B, it was confirmed that the 70% EtOH extract of the roots and aerial parts of Deodeok did not exhibit cytotoxicity.

Experimental Example 2: Analysis of the effect of bioactive compounds of Deodeok extract on inhibiting SARS-CoV-2 entry into cells

For the eight major compounds isolated from the 70% EtOH extract of Deodeok root (Figure 2), two SARS-CoV-2 entry pathways, two SARS-CoV-2 entry pathways, i.e., ACE2-positive (ACE2+) We sought to determine the inhibitory effect on the endolysosomal pathway of cells and the TMPRSS2-mediated direct membrane fusion pathway in ACE2 and TMPRSS2 double-positive (ACE2/TMPRSS2+) cells.

The results are shown in Figure 3. As a result of pSARS-CoV-2 entry assay, pentacyclic triterpenoid saponins such as lancemaside A (LA) and lancemaside B were activated in ACE2-only expressing cells (ACE2+). and H1299 cells (ACE2/TMPRSS2+) expressing both ACE2 and TMPRSS2 effectively inhibited the entry of SARS-CoV-2, while other compounds showed no significant effect (Figure 3A).

In particular, lancemicide A (LA) showed the strongest inhibitory activity in ACE2+ and ACE2/TMPRSS2+ H1299 cells, with IC _50s of 1.77 and 2.85 μM, respectively (Figure 3B).

Experimental Example 3: Analysis of the effect of inhibiting cell entry of the D614G mutation of lancemaside A (LA)

Among the S mutations, the D614G mutation is the basal B.1 lineage of SARS-CoV-2 and is the ancestor of the Delta variant, which is currently the greatest threat worldwide. The D614G mutation is the spike protein (spike protein). ) Amino acid number 614 of the receptor binding domain (RBD) is substituted from aspartic acid (Asp, D) to glycine (Gly, G). This mutation is known to increase the infectivity of SARS-CoV-2 by improving its binding affinity for ACE2. Similarly, pSARS-CoV-2 virus carrying the D614G substitution showed greater entry ability than the wild type (WT) virus in both ACE2+ and ACE2/TMPRSS2+ H1299 cells. It was about two times higher (A in Figure 4).

The effect of lancemicide A (LA) on the cellular entry of this D614G mutation was evaluated. The results are shown in Figure 4B, from which lancemaside A (LA) has IC ₅₀ values of 2.09 and 2.85 μM for ACE2+ and ACE2/TMPRSS2+ H1299 cells, respectively, and for both ACE2+ and ACE2/TMPRSS2+ H1299 cells. It was confirmed that mutant infection was suppressed by inhibiting the entry of mutant viruses. This was almost identical to that of the parental pSARS-CoV-2 virus (Figure 4B). In addition, it was confirmed that lancemaside A (LA) does not exhibit cytotoxicity against both ACE2+ and ACE2/TMPRSS2+ H1299 cells.

Experimental Example 4: Analysis of anti-SARS-CoV-2 activity of lancemicide A (LA)

The anti-SARS-CoV-2 activity of lancemicide A (LA) was analyzed using the infectious authentic SARS-CoV-2 virus.

As a result of evaluating SARS-CoV-2 infection based on immunostaining of SARS-CoV-2 nucleocapsid N protein, the IC ₅₀ of lancemicide A (LA) was 2.62 μM. , it was confirmed that it exhibits antiviral activity against infection by early SARS-CoV-2 isolate (lineage A) in Vero cells (Figure 5).

Additionally, lancemarside A (LA) can block infection with alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2), and omicron (B.1.1.529) variants. As a result of checking whether lancemicide A (LA) was found to have an IC ₅₀ of 3.37 μM, 3.12 μM, 2.23 μM, and 2.45 μM against each mutant virus, respectively, it was an equally powerful antibacterial agent against these SARS-CoV-2 variants. It was confirmed to have viral activity (Figure 6).

The above results suggest that LA can be a broad-spectrum antiviral agent that efficiently blocks infection with the SARS-CoV-2 parental strain and is also active against variants.

Experimental Example 5: Analysis of anti-SARS-CoV-2 activity of lancemicide A (LA)

We sought to determine the effect of lancemicide A (LA) on the process of virus fusion with the host cell membrane, which is the final step in the penetration of SARS-CoV-2 into host cells.

First, we established a monitoring system to visualize SARS-CoV-2 membrane fusion with host cells. This system was designed to stabilize the S protein and EGFP in a single bicistronic mRNA in HEK293T cells (S-HEK293T). Based on expression and labeling of ACE2/TMPRSS2+ H1299 cells combined with mCherry (Figure 7A).

A suspension of Spike-HEK293T cells was added to a monolayer of ACE2/TMPRSS2+ H1299 cells, and observation using time-lapse microscopy showed that Spike-HEK293 cells gradually approached and interacted with H1299 cells, and then formed two different cells. It was confirmed that different types of cells fused quickly. This fusion event caused the hybrid cells to display the S protein on their surface and subsequently fuse with neighboring H1299 cells to form enlarged multinucleated cells.

As a result of confirming the effect of lancemarside A (LA) on this fusion event, surprisingly, it was confirmed that treatment with lancemarside A (LA) almost completely blocked the fusion between two cells, preventing the formation of multinucleated cells ( Figure 7B).

As a result of quantitative analysis through flow cytometry, when ACE2/TMPRSS2+ H1299 cells and Spike-HEK293T cells were mixed, 31.1% of the cells achieved cell fusion, whereas when pretreated with lancemaside A (LA), this ratio was lower. It was confirmed that it dropped to 3.7% (C in Figure 7).

Multinucleated giant cells called syncytium are often detected in the lung tissue of patients infected with SARS-CoV-2, which is one of the clinical and pathological hallmarks of COVID-19.

Experimental Example 6: Analysis of syncytium formation inhibition ability of lancemicide A (LA)

To investigate the effect of lancemaside A (LA) on syncytium formation, split-GFP technology was used, in which half of GFP is expressed separately in two cells (Figure 8A). This reconstitutes functional fluorescent proteins upon cell-to-cell fusion, producing detectable GFP fluorescence.

ACE2/TMPRSS2+ H1299 cells expressing each of the two fragments of the reporter protein were generated and plated in equal numbers. The next day, this cell mixture was transduced with rentvirus to express the S protein for 6 hours, then treated with various concentrations of LA and cultured for an additional 24 hours (Figure 8B).

In DMSO-treated control cells, several enlarged GFP+ cells were detected, confirming that syncytium was formed upon S expression (Figure 8C). Additionally, DAPI nuclear staining showed that the nuclei were clustered to form nuclear aggregates in the center of the syncytium (Figure 8C). On the other hand, treatment with LA reduced the formation of nuclear clusters, confirming that LA inhibits syncytium formation (Figure 8C). On the other hand, treatment with LA reduced syncytium formation, which showed a LA concentration-dependent trend (Figure 8C and D). In addition, we observed that LA also inhibited the induction of apoptosis through caspase cascade activation induced by syncytium formation (Figure 8E). Through these experiments, it was confirmed that LA inhibits syncytium formation.

Experimental Example 7: Sagebrush ( Aster pseudoglehnii ), tuna ( Aster scaber ), Raw paper ( Polygala tenuifolia ) and beetle poison ( Aster koraiensis ) Analysis of the inhibitory effect of the extract on SARS-CoV-2 entry into cells

For each 70% EtOH extract of Aster pseudoglehnii , Aster scaber , Polygala tenuifolia and Aster koraiensis , SARS- A SARS-CoV-2 pseudovirus carrying the CoV-2 spike (S) protein was used to show an inhibitory effect on the entry of SARS-CoV-2 into cells as in Experimental Example 1 above. was measured, and the results are shown in Figure 9.

The pSARS-CoV-2 entry analysis results showed that each extract of Aster pseudoglehnii , Aster scaber , Polygala tenuifolia , and Aster koraiensis had two SARS-CoV-2 entry routes, namely , it was confirmed that it exhibited inhibitory activity against the endolysosomal pathway in cells expressing ACE2 alone (ACE2+) and the TMPRSS2-mediated direct membrane fusion pathway in H1299 cells expressing both ACE2 and TMPRSS2 (ACE2/TMPRSS2+).

Experimental Example 8: Bee ant taste ( Aster koraiensis ) Analysis of the inhibitory effect of the extract on SARS-CoV-2 entry into cells

A 70% EtOH extract from the leaves of A. koraiensis showed SARS-CoV-2-like HIV-based lentiviral particles (pSARS-CoV-2) carrying the SARS-CoV-2 spike (S) protein. A virus (SARS-CoV-2 pseudovirus) was used to determine whether it had an inhibitory effect on the entry of SARS-CoV-2 into cells as in Experimental Example 1 above. The results are shown in Figure 10.

The leaf extract of A. koraiensis showed strong anti-SARS-CoV-2 activity in ACE2+ and ACE/TMPRSS2+ H1299 cells, with an IC ₅₀ of 48.4 and 62.4 μg/ml, respectively, and was not cytotoxic up to 200 μg/ml. It was confirmed that this was not the case (Figure 10).

Experimental Example 9: Analysis of the effect of bioactive compounds of A. koraiensis extract on inhibiting SARS-CoV-2 entry into cells

For the two major compounds isolated from the 70% EtOH extract of A. koraiensis leaves (Figure 11A), two SARS-CoV-2 entry routes were identified, namely, endolysosomes in ACE2-positive (ACE2+) cells. The inhibitory effect on the endolysosomal pathway and the TMPRSS2-mediated direct membrane fusion pathway in ACE2 and TMPRSS2 double-positive (ACE2/TMPRSS2+) cells was confirmed, and the results are shown in Figure 11B.

pSARS-CoV-2 entry assay results show that pentacyclic triterpenoid saponins such as astersaponin I are effective against cells expressing ACE2 alone (ACE2+) and H1299 cells expressing both ACE2 and TMPRSS2 (ACE2 cells) without cytotoxicity. /TMPRSS2+) effectively blocked the entry of SARS-CoV-2. In particular, in the case of astersaponin I, IC ₅₀ was 1.60 and 2.89 μM in ACE2+ and ACE2/TMPRSS2+ H1299 cells, respectively.

Experimental Example 10: Analysis of syncytium formation inhibition ability of astersaponin I

To investigate the effect of astersaponin I on syncytium formation, split-GFP technology was used. The results are shown in Figure 12.

GFP-positive cells showing syncytia with aggregated DAPI-positive multiple nuclei were observed only when S-protein was overexpressed in ACE2/TMPRSS2+ expressing each fragment of split-GFP. On the other hand, treatment with astesaponin I (AI) effectively reduced the number of cells showing GFP fluorescence and nuclear aggregation in a dose-dependent manner (Figure 12A), with an IC _{50 of} 3.9 μM (Figure 12B). It was confirmed that astesaponin I (AI) effectively blocks S-mediated cell-to-cell fusion, that is, syncytium formation.

Experimental Example 11: Analysis of the intracellular entry inhibition effect of the D614G mutation of astesaponin I

The effect of astersaponin I on cellular entry of the D614G mutation was evaluated.

The results are shown in Figure 13, and it was confirmed that astersaponin I treatment inhibits the entry of the mutant virus into both ACE2+ and ACE2/TMPRSS2+ H1299 cells, respectively. IC ₅₀ values were 2.02 and 2.99 μM, which were similar to those of the WT virus (Figure 13).

Experimental Example 12: Analysis of anti-SARS-CoV-2 activity of astersaponin I

Astesaponin I is present in infectious authentic SARS-CoV-2 viruses, including early SARS-CoV-2 isolates (lineage A), as well as SARS-CoV-2 variants. It was confirmed whether it could broadly block infections.

As a result of evaluating SARS-CoV-2 infection based on immunostaining of SARS-CoV-2 nucleocapsid N protein, the astersaponin I IC ₅₀ was 1.94 μM. , it was confirmed that Vero cells exhibit antiviral activity against infection by early SARS-CoV-2 isolates (Ancestral SARS-CoV-2) (Figure 14, A and B).

Additionally, astersaponin I can block infection by alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2), and omicron (B.1.1.529) variants. As a result of checking whether it can be used, astersaponin I showed an IC ₅₀ of 2.04 μM, 1.94 μM, 2.04 μM, and 1.91 μM for each mutant virus, respectively, making it equally powerful against these SARS-CoV-2 variants. It was confirmed to have antiviral activity (Figure 14, A and B).

These results provide evidence that triterpenoid saponins with a specific structure can inhibit infection by SARS-CoV-2 variants and the parental strain regardless of S mutation status by blocking fusion of the host cell membrane and the viral membrane. This suggests the potential broad-spectrum anti-SARS-CoV-2 activity of triterpenoid saponins and suggests that it could be a broad-spectrum antiviral agent that is also active against SARS-CoV-2 variants.

That is, triterpenoid saponins with specific structures originating from plant extracts of Codonopsis , Aster , and/or Polygala inhibit the two entry pathways used by SARS-CoV-2. It can act as a natural viral membrane fusion blocker that can prevent SARS-CoV-2 original strains and variants from infecting host cells with similar efficiency, disrupting S-mediated cell-to-cell fusion and forming the syncytium. ) can inhibit the formation. In addition, triterpenoid saponins with a specific structure derived from extracts of Deodeok and Siberian antler can act as a broad-spectrum antiviral agent against not only SARS-CoV-2 but also other new enveloped viral pathogens that may arise in the future. .

As above, specific parts of the content of the present invention have been described in detail, and for those skilled in the art, it is clear that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. It will be obvious. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (17)

A pharmaceutical composition for preventing or treating enveloped virus infections, comprising a compound represented by the following formula (1).
[Formula 1]

In Formula 1,
R ¹ is hydrogen or hydroxy;
R ² is methyl or hydroxymethyl;
R ³ is

or

ego;
R ^a is hydrogen or

ego;
R ^b is hydrogen or

ego;
^Rc is

or

am. According to clause 1,
The compound of Formula 1 is a pharmaceutical composition for preventing or treating enveloped virus infections, which is represented by the following Formula 2.
[Formula 2]

In Formula 2, R ^a is hydrogen or

am. According to clause 1,
The compound of Formula 1 is a pharmaceutical composition for preventing or treating enveloped virus infections, which is represented by the following Formula 3.
[Formula 3]

According to clause 1,
The enveloped virus is selected from Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2), Middle East Respiratory Syndrome (MERS) virus, and Severe Acute Respiratory Syndrome (SARS) virus. A pharmaceutical composition for preventing or treating enveloped virus infections. According to clause 1,
The compound of Formula 1 is a pharmaceutical composition for preventing or treating enveloped virus infections, which inhibits cell entry of enveloped viruses and/or inhibits binding to spike proteins of enveloped viruses. According to clause 1,
A pharmaceutical composition for preventing or treating enveloped virus infections, wherein the compound of Formula 1 is contained in an amount of 0.001 to 50% by weight based on the total weight of the composition. A pharmaceutical composition for preventing or treating enveloped virus infections comprising Codonopsis extract, Aster extract, Polygala extract, or a mixture thereof. According to clause 7,
The enveloped virus is selected from Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2), Middle East Respiratory Syndrome (MERS) virus, and Severe Acute Respiratory Syndrome (SARS) virus. A pharmaceutical composition for preventing or treating enveloped virus infections. According to clause 7,
The Codonopsis extract, Aster extract, Polygala extract, or a mixture thereof inhibits cell entry of the enveloped virus and/or inhibits binding to the spike protein of the enveloped virus. , Pharmaceutical composition for preventing or treating enveloped virus infections. According to clause 7,
The Codonopsis extract, Aster extract, Polygala extract, or mixtures thereof include a compound represented by the following formula (1), which is used for prevention of enveloped virus infections or Pharmaceutical composition for therapeutic use.
[Formula 1]

In Formula 1,
R ¹ is hydrogen or hydroxy;
R ² is methyl or hydroxymethyl;
R ³ is

or

ego;
R ^a is hydrogen or

ego;
R ^b is hydrogen or

ego;
^Rc is

or

am. According to clause 10,
The compound of Formula 1 is a pharmaceutical composition for preventing or treating enveloped virus infections, which is represented by the following Formula 2.
[Formula 2]

In Formula 2, R ^a is hydrogen or

am. According to clause 10,
The compound of Formula 1 is a pharmaceutical composition for preventing or treating enveloped virus infections, which is represented by the following Formula 3.
[Formula 3]

According to clause 7,
Infection with enveloped viruses, wherein the Codonopsis extract, Aster extract, Polygala extract, or a mixture thereof is contained in an amount of 0.001 to 50% by weight based on the total weight of the composition. Pharmaceutical composition for prevention or treatment of. A pharmaceutical preparation comprising a pharmaceutical composition for preventing or treating enveloped virus infections according to any one of claims 1 to 13. According to clause 14,
The pharmaceutical preparation is a solid dosage form, a spray dosage form, or a liquid dosage form. According to clause 14,
The pharmaceutical preparation is for oral, parenteral, intranasal, oral, buccal, sublingual, airway spray, or rectal administration. According to clause 14,
The pharmaceutical preparation is in the form of a capsule, pill, tablet, solution, suspension, spray, foam, patch or paste.

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