Composition for treating corona virus comprising extract of Saururus chinensis

Abstract

The present invention relates to a composition for the prevention or treatment of coronavirus comprising an extract of Saururus chinensis, or saucerneol, saucernetin-8, or manassantin B isolated from Saururus chinensis. Through the present invention, pharmaceutical compositions, health functional food compositions, and feed compositions are provided that have low side effects and resistance and excellent antiviral activity against coronavirus.

Description

Technology Field

[0001] The present invention is Sambeakcho ( Saururus chinensis The present invention relates to a composition for the prevention or treatment of coronavirus comprising an extract. Specifically, the present invention relates to a composition comprising a *Sambaeckcho* extract having excellent antiviral activity against SARS-CoV-2, or saucerneol, saucernetin-8, or manassantin B isolated from the *Sambaeckcho* extract. Background Technology

[0003] Coronaviruses (CoVs) are a group of viruses belonging to the order Nidovirales, family Coronaviridae, and subfamily Orthocoronavirinae in phylogenetic classification. Based on their genetic structure, these coronaviruses can be subdivided into four groups: alpha coronavirus (α-coronavirus), beta coronavirus (β-coronavirus), gamma coronavirus (γ-coronavirus), and delta coronavirus (δ-coronavirus) (Yu Chen et al , 2020; Woo PC et al , 2012).

[0004] Human coronaviruses belonging to the alpha coronavirus family include human coronavirus-229E and human coronavirus-NL63, and human coronaviruses belonging to the beta coronavirus family include human coronavirus-OC43 and human coronavirus-HKU1 (Hamre D et al , 1966; Fouchier RA et al , 2004; Bucknall RA et al , 1972; Woo PC et al , 2005).

[0005] The four human coronaviruses mentioned above generally cause the seasonal common cold, which presents with mild respiratory symptoms. In contrast, human coronaviruses that cause severe acute respiratory infections in humans include the severe acute respiratory syndrome coronavirus (SARS-CoV), the Middle East respiratory syndrome coronavirus (MERS-CoV), and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; coronavirus disease 2019, COVID-19); all of these coronaviruses belong to the betacoronavirus group. The aforementioned severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as COVID-19, shares approximately 80% of its genetic sequence with SARS-CoV (Drosten C et al , 2003; Chan JF et al , 2015; Zhou P et al , 2020).

[0006] Encased in an envelope, the coronavirus is a virus measuring 80–220 nm in size with a positive-sense single-stranded RNA genome. It was named coronavirus because the 20 nm crown-like spikes on its envelope make it resemble the sun's corona in electron microscope images, and the coronavirus has the largest genome among currently known RNA viruses (Englund JA et al , 2019).

[0007] The nucleoprotein (N) of the coronavirus surrounds the RNA genome to form a coiled tubular helix, which is enclosed by a lipid-containing envelope (E). The coronavirus envelope contains two or three glycoproteins, such as matrix protein (M) embedded within the envelope, S protein protruding from the envelope surface in the form of crown-like spikes, and hemagglutinin esterase (HE), which is present only in some coronaviruses; infection begins when the aforementioned HE or S protein, known to act as a target for neutralizing antibodies, binds to a cell receptor. The coronavirus genome shares genes for five proteins, including the viral replication enzyme RNA-dependent RNA polymerase (RdRp), spike protein (S), envelope (E), matrix protein (M), and nucleoprotein (N), and these genes are arranged in the sequence 5′-RdRp-SEMN-3′ (Brian DA et al , 2005).

[0008] Human coronaviruses that infect humans—human coronavirus-229E, human coronavirus-NL63, human coronavirus-OC43, and human coronavirus-HKU1—generally cause mild upper respiratory disease and are associated with 10–30% of patients with the common cold. Coronaviruses, once known merely as one of the causes of the common cold, have formed various mutations starting with the SARS-CoV outbreak in 2002, through MERS-CoV, and currently reaching SARS-CoV-2 (COVID-19), causing novel infectious diseases such as severe infectious pneumonia in humans (Paules CI et al , 2020; Weiss SR et al , 2011; Fehr AR et al, 2015).

[0009] Although various vaccines and treatments to overcome SARS-CoV-2 are currently being developed, there is still a lack of established treatments. Furthermore, as the epidemic continues, the virus mutates, changing the transmission and severity of the disease, which is affecting the effectiveness of clinically used vaccines and treatments (World Health Organization, "Tracking SARS-CoV-2 variants").

[0010] Although various antiviral agents are currently being developed and used, RNA viruses present problems such as increasing viral resistance to antiviral drugs; therefore, active efforts are underway to develop antiviral agents that are more effective, safe, and can be produced at a lower cost. However, despite these efforts, there are still no safe antiviral agents that demonstrate effective therapeutic effects against RNA viruses (SA Jassim et al , 2003).

[0011] Medicinal plants often exhibit antiviral effects against various viruses and have the advantages of low production costs, low potential for side effects and resistance. Until recently, it has been verified that many medicinal plants demonstrate potent antiviral effects when applied to humans or animals infected with viruses (PS Venkateswaran et al , 1987; SP Thyagarajan et al (1988). For this reason, many scientists around the world, including in Asia, Europe, and the United States, are striving to develop antiviral agents from traditional medicine based on medicinal plants (MM Cowan, 1999; DP Briskin, 2000; JE Williams, 2001).

[0012] Meanwhile, Sambeakcho ( Saururus chinensisIt is a medicinal plant that has been used to treat hypertension, pneumonia, edema, jaundice, etc., and is also used to treat various inflammatory diseases (Rao KV et al , 1990; Jeong HJ et al , 2015). The roots of *Sambaeckcho* contain various types of lignans, such as saucerneol, di-O-methyltetrahydrofuriguaiacin B, machilin D, sauchinone, and manassantin (Li C et al , 2017; Seo CS, Lee YK et al , 2008; Seo CS, Zheng MS et al , 2008; Sung SH, 2006), some of them exhibit anti-inflammatory or antioxidant effects.

[0013] The inventors completed the present invention by discovering that the extract of *Sambaeckcho* and Saucerneol, Saucernetin-8, and Manassantin B isolated from *Sambaeckcho* possess antiviral activity against SARS-CoV-2, which was previously unknown. The problem to be solved

[0015] The present invention aims to provide a pharmaceutical composition for the prevention or treatment of coronavirus, and in particular, to provide a pharmaceutical composition that is highly safe and has excellent antiviral activity against SARS-CoV-2, and furthermore, to provide a health functional food composition and an animal feed composition for the prevention or improvement of SARS-CoV-2 infection. means of solving the problem

[0017] For the prevention or treatment of coronavirus infection, the present invention relates to *Sambaeckcho* ( Saururus chinensis Provides a pharmaceutical composition comprising an extract as an active ingredient.

[0018] In one embodiment, the pharmaceutical composition may be for the prevention or treatment of coronavirus infection, and preferably for the prevention or treatment of SARS-CoV-2 infection.

[0019] In one embodiment, the extract may be extracted using water, a C1 to C4 lower alcohol, or a mixture thereof as a solvent, preferably using a C1 to C4 lower alcohol as a solvent, and more preferably using methanol as a solvent.

[0020] In addition, the present invention provides a pharmaceutical composition comprising, as an active ingredient, one or more compounds selected from saucerneol, saucernetin-8, and manassantin B, or a pharmaceutically acceptable salt thereof.

[0021] In one embodiment, the pharmaceutical composition may be for the prevention or treatment of coronavirus infection, and preferably for the prevention or treatment of SARS-CoV-2 infection.

[0022] In one embodiment, the saucerneol, saucernetin-8, or manassantin B may be extracted from *Sambaeckcho*, and preferably obtained from an ethyl acetate fraction fractionated by adding an ethyl acetate solvent to a methanol extract of *Sambaeckcho*.

[0023] In addition, the present invention (Sambaeckcho ( Saururus chinensis Provides a food composition for preventing or improving SARS-Coronavirus-2 infection comprising an extract as an active ingredient.

[0024] In addition, the present invention provides an animal feed composition for the prevention or improvement of SARS-CoV-2 infection comprising a *Sambucus nigra* extract as an active ingredient.

[0025] In addition, the present invention provides a food composition for preventing or improving SARS-CoV-2 infection comprising one or more compounds selected from saucerneol, saucernetin-8, and manassantin B, or a pharmaceutically acceptable salt thereof.

[0026] In addition, the present invention provides an animal feed composition for the prevention or improvement of SARS-CoV-2 infection comprising one or more compounds selected from saucerneol, saucernetin-8, and manassantin B, or a pharmaceutically acceptable salt thereof. Effects of the invention

[0028] Saucerneol, saucernetin-8, and manassantin B, compounds isolated from the methanol extract of *Sambaeckcho* and its ethyl acetate fraction according to the present invention, possess excellent antiviral activity against coronaviruses and can be particularly useful for the prevention or treatment of SARS-CoV-2 infection. Furthermore, they can be utilized as health functional food compositions and animal feed compositions for the prevention or improvement of SARS-CoV-2 infection. In particular, the extracts and active ingredients of the present invention have low side effects and resistance, and relatively low production costs, making them highly industrially applicable. Brief explanation of the drawing

[0030] Figure 1 shows the experimental results of measuring the antiviral activity of the methanol extract of *Sambaeckcho* against SARS-CoV-2. Figures 2 to 4 show the experimental results of measuring the antiviral activity against SARS-CoV-2 of saucerneol, saucernetin-8, and manassantin B, which are active ingredients derived from *Sambaeckcho*. Figure 5 shows a phase-contrast microscope image of the effects of saucerneol, saucernetin-8, and manassantin B, which are active ingredients derived from *Sambekcho*, on the cell viability of cells infected with SARS-CoV-2. Figures 6 to 8 show the experimental results of measuring the antiviral activity of saucerneol, saucernetin-8, and manassantin B against SARS-CoV-2 through Real-time PCR analysis. Specific details for implementing the invention

[0031] The present invention is Sambeakcho ( Saururus chinensis Provides a pharmaceutical composition comprising an extract as an active ingredient.

[0032] In addition, the present invention provides a pharmaceutical composition comprising one or more of the compounds saucerneol, saucernetin-8, and manassantin B isolated from a fraction of a *Sambeakcho* extract, or a pharmaceutically acceptable salt thereof.

[0033] Hereinafter, embodiments and examples of the present invention will be described in detail with reference to the attached drawings so that those skilled in the art can easily implement the present invention. However, the present invention may be embodied in various forms and is not limited to the embodiments and examples described herein.

[0034] Throughout this specification, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0035] The term "extract" as used herein has the meaning commonly understood in the art as a crude extract, but in a broader sense, it also includes fractions obtained by further fractionating the extract. That is, the extract includes not only that which is obtained using an extraction solvent, but also that which is obtained by additionally applying a purification process thereto. For example, fractions obtained through various additional purification methods, such as a fraction obtained by passing the extract through an ultrafiltration membrane having a specific molecular weight cut-off value, or separation by various chromatographs (designed for separation based on size, charge, hydrophobicity, or affinity), are also included in the extract of the present invention. Furthermore, the extract may be in the form of a solution (flowing extract), a concentrate (soft extract), or a dry powder, but is not limited thereto.

[0036] The extract used in the present invention can be obtained using a conventional extraction solvent known in the art. Polar solvents or non-polar solvents may be used as extraction solvents. Polar solvents include water, C1 to C6 alcohols (e.g., methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, and normal-butanol, etc.), acetic acid, or mixtures of the above polar solvents. Non-polar solvents include acetone, acetonitrile, ethyl acetate, methyl acetate, butyl acetate, fluoroalkanes, hexane, ether, chloroform, dichloromethane, or mixtures of the above non-polar solvents.

[0037] The extract used in the present invention may be extracted through hot water extraction, immersion (cold immersion or hot immersion) extraction, reflux cooling extraction, ultrasonic extraction, or conventional extraction methods known in the art.

[0038] As used herein, the term "fraction" refers to a result obtained by performing fractionation to separate a specific component or a specific group of components from a mixture containing various constituent components. In the present invention, the fractionation method for obtaining said fraction is not particularly limited and may be performed according to methods commonly used in the art.

[0039] As used herein, the term "active ingredient" means a component that exhibits the intended activity alone or can exhibit activity in combination with a carrier that is inactive itself.

[0040] As used herein, the term "prevention" refers to any act of suppressing or delaying the onset of a disease by administering the composition; "treatment" refers to any act of improving or beneficially altering the symptoms of an individual suspected of or suffering from the disease by administering the composition; and "improvement" refers to any act of at least reducing parameters related to the condition, such as the degree of symptoms, by administering the composition.

[0041] The animal feed composition of the present invention can be fed to all non-human animals, for example, non-human primates, sheep, dogs, cattle, horses, etc.

[0042] The saucerneol used in the present invention has the structure of the following chemical formula I.

[0043] [Chemical Formula I]

[0044]

[0046] Saucernetin-8 used in the present invention has the structure of the following chemical formula II.

[0047] [Chemical Formula II]

[0048]

[0050] The manassantin B used in the present invention has the structure of the following chemical formula III.

[0051] [Chemical Formula III]

[0052]

[0054] The present invention is to be explained in more detail through the following examples, but the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0056] [Example 1]

[0057] Separation of Trianthema portulacastrum extract, fractions thereof, and active ingredients

[0059] 1.2 kg of *Sambaeckcho* was extracted with 100% methanol for 72 hours, and the methanol extract was separated using hexane, ethyl acetate, and butanol. Subsequently, the antiviral activity against SARS-CoV-2 was measured using the separated hexane, ethyl acetate, and butanol fractions.

[0060] As a result, it was confirmed that the ethyl acetate layer exhibited high antiviral activity, and high-speed liquid chromatography (HPLC) was performed to separate saucerneol, saucernetin-8, and manassantin B from the ethyl acetate layer.

[0062] [Example 2]

[0063] Evaluation of the antiviral activity of *Sambaeckcho* extract and active ingredients derived from *Sambaeckcho* against SARS-CoV-2

[0065] 2-1. Antiviral Activity Test

[0067] The antiviral activity of the *Sambaeckcho* extract and active ingredients obtained in Example 1 was evaluated as follows. The degree of reduction in cytotoxicity caused by SARS-CoV-2 infection was evaluated by measuring the total amount of protein in surviving cells as absorbance using the Sulforhodamine B assay (SRB assay) and calculating the result.

[0068] In addition, to evaluate antiviral activity against SARS-CoV-2, SARS-CoV-2 (NCCP NO. 43382) was obtained from the National Human Bioresource Bank of the Korea Disease Control and Prevention Agency, and the virus was amplified in Vero-E6 cells at 37°C. Vero-E6 cells were cultured in DMEM media supplemented with 10% FBS and 0.01% antibiotics.

[0069] Subsequently, cells susceptible to SARS-CoV-2 were placed in 3 x 10 wells of a 96-well culture plate (Nunc, Denmark). 4 The cells were prepared in the specified number and cultured for 24 hours until they had grown to about 90% capacity in a 96-well culture plate. After removing the medium from each well, the cells were washed once with PBS, and 10 μL of methanol extract of *Sambaeckcho*, saucerneol, saucernetin-8, and manassantin B were added to 90 μL of DMEM containing 1% FBS and SARS-CoV-2 at three concentrations: 0.4 μg / mL, 2 μg / mL, and 10 μg / mL.

[0070] The methanol extract of *Sambaeckcho*, saucerneol, saucernetin-8, and manassantin B were dissolved in DMSO and diluted in MEM media. The final concentration of DMSO was set to 0.1%, which has no effect on cell culture, and 0.1% DMSO was used as a cell control.

[0071] Four wells were used as a control group treated only with the virus without the compound, and another four wells were used as a control group treated with neither the virus nor the compound. After culturing for 2 days in a CO2 incubator (VS-9108MS, Vision, Korea) at 37°C and 5% CO2, the cell morphology was observed at 32 x 10x magnification using a phase-contrast microscope (Axiovert 10; Zeiss, Wetzlar, Germany) and images were recorded.

[0073] 2-2. Measurement of Antiviral Activity

[0075] The antiviral activities of the methanol extract of *Sambaeckcho*, saucerneol, saucernetin-8, and manassantin B were measured using the above antiviral activity test method, and the results are shown in Table 1 below. In Table 1 below, CC 50 (Cytotoxic concentration 50%) is a value used to confirm the cytotoxic effect, representing the concentration of a compound required to reduce cell viability by 50%, and IC 50 (Inhibitory concentration 50) is a value used to confirm antiviral effects, representing the concentration that inhibits viral activity by 50%, and TI (Therapeutic index) is a therapeutic index, a measure of drug safety, and the higher the value, the greater the difference between the effective dose and the toxic dose, meaning that even if used for treatment, there are fewer side effects.

[0077] Antiviral activity against SARS-CoV-2 compound CC 50 (μg / mL) IC 50 (μg / mL) TI methanol extract 50 < 0.76 ± 0.28 65.78 Saucerneol 1.22 ± 0.16 40.98 Saucernetin-8 2.30 ± 0.73 21.73 Manasantine B 4.16 ± 1.04 12.01

[0078] As shown in Table 1 above, when Vero-E6 infected with SARS-CoV-2 was treated with the methanol extract of *Sambaeckcho*, the IC50 50The value is 0.76 μg / mL, and the IC50 is when treated with saucerneol, saucernetin-8, and manassantin B. 50 The values ​​were 1.22 μg / mL, 2.3 μg / mL, and 4.16 μg / mL, respectively, showing excellent antiviral activity against SARS-CoV-2.

[0079] The TI values ​​related to drug safety were found to be 65.78, 40.98, 21.73, and 12.01 for the methanol extract of *Sambaeckcho*, saucerneol, saucernetin-8, and manassantin B, respectively.

[0080] Therefore, the methanol extract of *Sambaeckcho* and the active ingredients derived from *Sambaeckcho*, namely saucerneol, saucernetin-8, and manassantin B, exhibited excellent stability.

[0082] 2-3. Measurement of Cell Viability

[0084] To evaluate antiviral activity through cell viability analysis, the cell viability of Vero-E6 cells infected with SARS-CoV-2 was analyzed using an SRB assay. Methanol extract of *Sambaeckcho* (ST), saucerneol, saucernetin-8, and manassantin B were added to Vero-E6 cells infected with SARS-CoV-2 at three concentrations: 0.4 μg / mL, 2 μg / mL, and 10 μg / mL, and the results are shown in Figures 1 to 4.

[0085] As a result of analyzing the cell viability of Vero-E6 cells infected with SARS-CoV-2, all groups treated with methanol extract of *Sambaeckcho*, saucerneol, saucernetin-8, and manassantin B at a concentration of 10 μg / mL showed a cell viability of over 70%, which was superior compared to the cell viability of Vero-E6 cells (Veh) infected with SARS-CoV-2 (approx. 35%).

[0086] In addition, cell viability was confirmed using a phase-contrast microscope after treatment with 10 μg / mL of saucerneol, saucernetin-8, and manassantin B. As shown in Figure 5, it was confirmed that more cells survived when treated with the active ingredients compared to a control group treated only with the virus.

[0087] Therefore, through the above experimental results, it can be confirmed that the methanol extract of *Sambaeckcho* and its active ingredients, saucerneol, saucernetin-8, and manassantin B, have excellent antiviral effects against SARS-CoV-2.

[0089] [Example 3]

[0090] Antiviral activity analysis via Real-time PCR

[0092] Real-time PCR was performed to measure the antiviral activity against SARS-CoV-2 of saucerneol, saucernetin-8, and manassantin B, which are active ingredients isolated from *Sambaeckcho*. Vero E6 cells were infected with SARS-CoV-2, and then treated with saucerneol, saucernetin-8, and manassantin B at concentrations of 0.4 μg / mL, 2 μg / mL, and 10 μg / mL.

[0093] After 24 hours had passed since the above active ingredient was treated, viral RNA was extracted using QIAcube (Qiagen, Germany), an automated RNA extraction device from Qiagen, and real-time PCR was performed using PowerChek™ SARS-CoV-2 Real-time PCR Kit (IR6902) from Kogen. The E gene and ORF1 ab gene were detected using the Real-time PCR kit, and the SARS-CoV-2 E gene and ORF1 ab gene were detected using qRT-PCR.

[0094] The PCR reaction conditions were heated at 50°C for 30 minutes for reverse transcription, reverse transcription inactivation and initial denaturation at 95°C for 10 minutes, followed by a total of 40 cycles of 15 seconds at 95°C and 1 minute at 60°C, and the PCR values ​​were normalized to GAPDH values ​​and are shown in Figures 6 to 8.

[0095] As a result of treating with manasantine B, saucerneol, and saucernetin-8 for 24 hours after SARS-CoV-2 infection, manasantine B inhibited the amplification of the SARS-CoV-2 E gene and ORF1 gene at a concentration of 10 μg / mL or higher, and saucerneol and saucernetin-8 inhibited the amplification of the SARS-CoV-2 E gene and ORF1 gene at a concentration of 2 μg / mL or higher.

[0096] Therefore, it was confirmed that saucerneol, saucernetin-8, and manasantin B, active ingredients isolated from *Sambaeckcho*, exhibit potent antiviral activity.

Claims

Claim 1 delete Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 A pharmaceutical composition for the prevention or treatment of SARS-CoV-2 infection comprising one or more compounds selected from saucerneol, saucernetin-8, and manassantin B, or a pharmaceutically acceptable salt thereof. Claim 6 delete Claim 7 A pharmaceutical composition according to claim 5, characterized in that the above-mentioned saucerneol, saucernetin-8, or manassantin B is extracted from *Sambaeckcho*. Claim 8 A pharmaceutical composition according to claim 5, wherein the saucerneol, saucernetin-8, or manassantin B is obtained from an ethyl acetate fraction fractionated by adding an ethyl acetate solvent to a methanol extract of *Sambaeckcho*. Claim 9 delete Claim 10 delete Claim 11 A food composition for the prevention or improvement of SARS-CoV-2 infection comprising one or more compounds of saucerneol, saucernetin-8, and manassantin B, or a pharmaceutically acceptable salt thereof. Claim 12 An animal feed composition for the prevention or improvement of SARS-CoV-2 infection comprising one or more compounds selected from saucerneol, saucernetin-8, and manassantin B, or a pharmaceutically acceptable salt thereof.

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