Use of a herbal extract for preparing a pharmaceutical composition for inhibiting an individual from being infected with novel coronavirus

Abstract

The present invention provides the use of a herbal extract for preparing a pharmaceutical composition for inhibiting a subject from being infected with the novel coronavirus, wherein the herbal extract comprises an alcohol extract of a Menispermaceae herb, and the Menispermaceae herb refers to Stephania sasakii or Stephania tetrandra.

Description

Technical Field

[0001] The present invention relates to the use of an herbal alcohol extract for preparing a pharmaceutical composition for inhibiting an individual from being infected with the novel coronavirus (SARS-CoV-2), and particularly to the use of an alcohol extract of an herb of the Menispermaceae family for preparing a pharmaceutical composition for inhibiting an individual from being infected with the novel coronavirus. Background Art

[0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus discovered in 2019 and belongs to the genus Betacoronavirus in the Coronaviridae family. This virus is an enveloped positive-sense single-stranded RNA virus with a genome length of approximately 29.7 kb, and it is the seventh known coronavirus that can infect humans. The main transmission route of this virus is the respiratory tract. It enters cells by binding the spike protein on the viral particle membrane to angiotensin-converting enzyme 2 (ACE2) on the host cell, and can infect human organs where ACE2 is widely distributed, including the lungs, heart, and kidneys.

[0003] SARS-CoV-2 has extremely high transmissibility. When it enters the respiratory tract and infects lung tissue, it will induce an inflammatory response, causing damage to the lung tissue, forming coronavirus disease 2019 (COVID-19), and ultimately progressing to acute respiratory distress syndrome with a high fatality rate. COVID-19 has become a global pandemic disease. Currently, the whole world is actively researching and developing new drugs against COVID-19, and blocking the binding of the spike protein to ACE2 is an important research and development strategy for anti-SARS-CoV-2 drugs.

[0004] Natural compounds have the advantages of rich sources and diverse skeletons, which are important bases for drug development. From 1981 to 2019, nearly half of the newly approved drugs by the US Food and Drug Administration were from natural products or their derivatives, such as narcotic drugs derived from cocaine, analgesics derived from morphine, vincristine, doxorubicin, and paclitaxel used to treat cancer, and penicillin derived from fungi used as an antibiotic, etc. Therefore, the present invention actively studies which natural compounds have the potential to be developed into new drugs against COVID-19. Summary of the Invention

[0005] The present invention relates to the use of an herbal extract for preparing a pharmaceutical composition for inhibiting an individual from being infected with the novel coronavirus (SARS-CoV-2), wherein the herbal extract includes an alcohol extract of an herb of the Menispermaceae family.

[0006] After research, it was found that the alkaloid compounds contained in Stephania tetrandra and Stephania cephalantha of the Menispermaceae family have the ability to inhibit virus infection and anti-inflammatory effects.

[0007] In the present invention, the herbal alcohol extract is an ethanol extract.

[0008] Preferably, the herbal alcohol extract is an alkaloid concentrated layer extract of the ethanol extract.

[0009] In one embodiment, the herbal alcohol extract is a bis-benzylisoquinoline alkaloid.

[0010] In a more preferred embodiment, the herbal alcohol extract refers to fangchinoline, cepharanoline or cycleanine.

[0011] In a more preferred embodiment, the herbal alcohol extract of the present invention inhibits the infection of the individual by the novel coronavirus by blocking the binding of the spike protein on the novel coronavirus to the angiotensin-converting enzyme 2 on the individual cell.

[0012] The novel coronavirus referred to in the present invention refers to the D614G variant or the Delta variant. More preferably, the novel coronavirus referred to in the present invention especially refers to the Delta variant. Most preferably, the novel coronavirus referred to in the present invention especially refers to the Omicron variant.

[0013] In the present invention, the individual referred to in the present invention is a human or a mammal. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Showing the effect of pseudolentivirus on the viability of ACE2-293T cells.

[0015] Figure 2 Shown is the effect of each alkaloid compound in the present invention on the infectivity of the Omicron variant S protein pseudolentivirus.

[0016] Figure 3 Showing the effect of each alkaloid compound in the present invention on the viability of ACE2-293T cells. DETAILED DESCRIPTION OF THE INVENTION

[0017] It should be understood that the detailed description of the embodiments is for explaining the preferred embodiments of the present invention, rather than intending to limit the present invention to certain embodiments. It should be noted that the present invention is intended to cover all alternative embodiments within the same spirit and scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0018] Natural compound

[0019] The present invention uses a screening system combining computer molecular simulation calculations and pseudolentiviruses to preliminarily screen from a library of 1300 natural compounds by calculating the affinity degree of the compounds with the spike protein receptor-binding region through virtual screening. Then, pseudolentivirus experiments are used to test the ability of the compounds to inhibit spike protein-mediated virus infection. The present invention preliminarily discovers that the alkaloid compounds rich in Stephania cephalantha Hayata and Stephania tetrandra S. Moore in the Menispermaceae family have the ability to inhibit virus infection.

[0020] After Stephania cephalantha Hayata is extracted with ethanol, chemical composition analysis and activity evaluation are carried out. Subsequently, in the alkaloid concentrated layer of the ethanol extract, in addition to fangchinoline, Stephania cephalantha Hayata also contains cepharanoline or cycleanine. These compounds all belong to bis-benzylisoquinoline alkaloids.

[0021] Pseudolentivirus

[0022] The VSV-G pseudolentiviruses (clone names: S3w.Fluc.Ppuro) of the control group and the VSV-G pseudolentiviruses expressing the spike protein of SARS-CoV-2 (clone names: nCoV-S-Luc-D614G; nCoV-S-Luc-B.1.617.2 and nCoV-S-Luc-B.1.1.529) used in the present invention are purchased from the RNAiCore core facility. Among them, the nCoV-S-Luc-D614G pseudolentivirus expresses the spike protein of the SARS-CoV-2 D614G variant (hereinafter referred to as the D614G S protein), the nCoV-S-Luc-B.1.617.2 pseudolentivirus expresses the spike protein of the SARS-CoV-2 Delta variant (hereinafter referred to as the Delta S protein), and the nCoV-S-Luc-B.1.1.529 pseudolentivirus expresses the spike protein of the SARS-CoV-2 Omicron variant (hereinafter referred to as the Omicron S protein).

[0023] Pseudolentivirus infection test

[0024] First, HEK-293T cells overexpressing the hACE-2 protein (hereinafter referred to as ACE2-293T cells) were seeded at 1×10 4 cells per well in a 96-well cell culture plate and cultured in an incubator at 37°C and 5% CO 2 .

[0025] The ACE2-293T cells were divided into a negative control group (untreated), a vehicle group (given HBSS), a D614G S protein group (given 0.5 R.I.U. of D614G S protein pseudolenti virus), a Delta S protein group (given 0.5 R.I.U. of Delta S protein pseudolenti virus), and an Omicron S protein group (given 0.5 R.I.U. of Omicron S protein pseudolenti virus).

[0026] Pseudolenti virus with 0.5 relative infection unit (R.I.U.) (0.5 R.I.U. / cell; i.e., 5×10 3 R.I.U. / well) was pretreated with antiviral agents at different concentrations in DMEM medium containing 10% fetal bovine serum (FBS) at 37°C for 1 hour. Subsequently, the medium of the ACE2-293T cells in the D614G S protein group, Delta S protein group, and Omicron S protein group was replaced with the pretreated pseudolenti virus solution and cultured for 24 hours. The vehicle group (given PBS) was treated with the same procedure by replacing the virus solution with PBS.

[0027] The infectivity of the VSVG, D614G S, Delta S, and Omicron S pseudolenti virus solutions was 2682 R.I.U. / μL, 737 R.I.U. / μL, 190 R.I.U. / μL, and 1070 R.I.U. / μL, respectively. After infecting host cells, the luciferase activity was measured using a luciferase assay system (E2520, Promega) and recorded by a fluorescence analyzer. Briefly, the ACE2-293T cells were used to measure the luciferase activity on the cell lysate using a luciferase substrate according to the manufacturer's instructions.

[0028] Pseudolenti-virus cytotoxicity assay

[0029] The ACE2-293T cells of the negative control group (untreated), the vehicle group (given HBSS), the D614G S protein group (given 0.5 R.I.U. of D614G S protein pseudolenti virus), the Delta S protein group (given 0.5 R.I.U. of Delta S protein pseudolenti virus), and the Omicron S protein group (given 0.5 R.I.U. of Omicron S protein pseudolenti virus) were tested for cell viability 24 hours after treatment. The results are shown in Table 1 and Figure 1 as shown, there were no significant differences in the cell viability of each group.

[0030] Table 1

[0031]

[0032] Data are expressed as mean ± standard error (n = 3)

[0033] Compared with the negative control group

[0034] Virus infection inhibition test

[0035] First, the present invention uses pseudolenti virus experiments to test whether Stephania cepharantha (SC) and Stephania tetrandra (ST) can inhibit virus infection mediated by the spike protein of SARS-CoV-2 and evaluate the spike proteins of different variants.

[0036] After extraction of Stephania cepharantha (SC) and Stephania tetrandra (ST) with ethanol, the alkaloid concentrated layer was further separated, and then the ethanol extracts and alkaloid concentrated layers of Stephania cepharantha (SC) and Stephania tetrandra (ST) were formulated into high-concentration drug solutions (1, 3, 10, and 30 mg / mL) with dimethyl sulfoxide (DMSO). The virus solution was added to 10% DMEM medium to prepare a virus solution with a concentration of 5×10 4 R.I.U. / mL. Take 1 mL of the virus solution and add 1 μL of DMSO or the drug solution at the specified concentration, and allow the virus and the extract to act at 37°C for 1 hour. ACE2-293T cells were seeded in 96-well plates at a cell density of 1×10 4 cells / well and cultured in DMEM containing 10% FBS and 10 μg / mL blasticidin at 37°C. After culturing for 10 hours to allow the cells to adhere, 100 μL of the mixture of the drug and the virus was added for cell infection. After culturing at 37°C for 24 hours, the luciferase activity in the 96-well plates was quantified according to the manufacturer's instructions to present the degree of virus infection.

[0037] The results are shown in Table 2. The half-maximal inhibitory (IC 50 ) concentrations of Stephania cephalantha Hayata ethanol extract (SC-EtOH) against the D614G S protein group, Delta S protein group, and Omicron S protein group were 24.64 ± 0.21 μg / mL, 16.01 ± 1.22 μg / mL, and 13.20 ± 1.63 μg / mL, respectively. The half-maximal inhibitory (IC 50 ) concentrations of Stephania tetrandra S. Moore ethanol extract (ST-EtOH) against the D614G S protein group, Delta S protein group, and Omicron S protein group were 11.64 ± 1.81 μg / mL, 16.06 ± 0.60 μg / mL, and 8.29 ± 1.50 μg / mL, respectively. Notably, the half-maximal inhibitory (IC 50 ) concentrations of the alkaloid-enriched layer of Stephania cephalantha Hayata ethanol extract (SC-EtOH-alkaloid) against the D614G S protein group, Delta S protein group, and Omicron S protein group were 3.79 ± 0.06 μg / mL, 4.44 ± 0.03 μg / mL, and 1.20 ± 0.17 μg / mL, respectively. The half-maximal inhibitory (IC 50 ) concentrations of the alkaloid-enriched layer of Stephania tetrandra S. Moore ethanol extract (ST-EtOH-alkaloid) against the D614G S protein group, Delta S protein group, and Omicron S protein group were 1.40 ± 0.04 μg / mL, 1.57 ± 0.01 μg / mL, and 0.55 ± 0.03 μg / mL, respectively. From these results, it can be seen that the main components of Stephania cephalantha Hayata and Stephania tetrandra S. Moore that inhibit virus infection mediated by the SARS-CoV-2 spike protein are present in the alkaloids.

[0038] Table 2

[0039]

[0040]

[0041] Concentration required for half-maximal inhibition (IC 50 , μg / mL), n = 3

[0042] To further explore which alkaloid compounds have the strongest ability to inhibit virus infection mediated by the SARS-CoV-2 spike protein, the present invention conducted subsequent studies using Stephania cephalantha Hayata.

[0043] After the present invention extracts Stephania sasakii Hayata with ethanol, chemical component analysis and activity evaluation are carried out. Subsequently, fangchinoline is found in the alkaloid concentrated layer of the ethanol extract. In addition, Stephania sasakii Hayata also contains cepharanoline or cycleanine. All of the above several compounds have the ability to inhibit virus infection.

[0044] Prepare DMSO solutions with different concentrations of the compounds fangchinoline, cepharanoline, and cycleanine using DMSO. Virus solution preparation: Add the virus solution to 10% DMEM to make the virus concentration 5×10 4 R.I.U. / mL. Take 1 mL of the virus solution and add 1 μL of DMSO or the DMSO solution of the designated compound. Let the virus and the drug act under the conditions of 37 °C for 1 hour. Seed ACE2-293T cells in a 96-well plate with a cell density of 1×10 4 cells / well. Culture them in DMEM containing 10% FBS and 10 μg / mL blasticidin at 37 °C. After culturing for 10 hours to allow the cells to adhere, add 100 μL of the mixture of the drug and the virus for cell infection. After culturing at 37 °C for 24 hours, the activities of these compounds against the D614G S protein group, the Delta S protein group, and the Omicron S protein group are quantified for luciferase activity in a 96-well plate according to the instructions of the manufacturer's manual to test the luciferase activity in the cells, so as to present the degree of virus infection. The calculation method is to take the luciferase activity reading of the control group as 100%, and subtract the luciferase activity reading of the control group from the luciferase activity reading of each virus variant S protein group as the judgment of the infection degree. The results are shown in Table 3.

[0045] Table 3

[0046]

[0047] Taking the Omicron variant S protein pseudolentivirus as a representative, it can be seen that the infection inhibition trends of each compound against the Omicron variant S protein pseudolentivirus ( Figure 2 ).

[0048] Further calculate the half-maximal inhibitory concentration (IC 50 ) of its antiviral S protein. The results are shown in Table 4. The E-64 compound group (a compound that is considered in existing research to be able to inhibit SARS-CoV-2 infection; the control group) for the D614G S protein group, the Delta S protein group, and the anti-Omicron S protein group's IC50 They were 23.12 ± 0.63 μM, 23.06 ± 1.30 μM, and 22.69 ± 1.28 μM, respectively.

[0049] Table 4

[0050]

[0051] The concentration required for 50% inhibition (IC 50 , μM), n = 3

[0052] Compared with the E-64 compound group, the dose of fangchinoline for 50% inhibition (IC 50 ) of the D614G S protein group was 4.64 ± 0.96 μM, the dose of fangchinoline for 50% inhibition (IC 50 ) of the Delta S protein group was 1.82 ± 0.02 μM, and the dose of fangchinoline for 50% inhibition (IC 50 ) of the Omicron S protein group was 1.44 ± 0.06 μM; the dose of cepharanoline for 50% inhibition (IC 50 ) of the D614G S protein group was 0.48 ± 0.09 μM, the dose of cepharanoline for 50% inhibition (IC 50 ) of the Delta S protein group was 1.64 ± 0.18 μM, and the dose of cepharanoline for 50% inhibition (IC 50 ) of the Omicron S protein group was 1.31 ± 0.07 μM; the dose of cycleanine for 50% inhibition (IC 50 ) of the D614G S protein group was 0.23 ± 0.02 μM, the dose of cycleanine for 50% inhibition (IC 50 ) of the Delta S protein group was 2.21 ± 0.50 μM, and the dose of cycleanine for 50% inhibition (IC 50 ) of the Omicron S protein group was 0.53 ± 0.03 μM.

[0053] From these results, it can be known that fangchinoline, cepharanoline, and cycleanine can all broadly inhibit virus infections mediated by the spike proteins of different SARS-CoV-2 variants.

[0054] Alkaloid cytotoxicity analysis

[0055] ACE2-293T was seeded into 96-well plates, with 1 × 10 4 cells in each well, at 37 °C, 5% CO 2Cultivate for 10 hours. Prepare 10 mM fangchinoline, cepharanoline, and cycleanine DMSO solutions. Take 1 mL of 10% DMEM medium without phenol red indicator and add 1 μL of DMSO or 10 mM alkaloid drug DMSO solution to prepare a one-thousandth diluted drug DMEM culture medium. After removing the cell culture medium of ACE2-293T, add 100 μL of the drug-containing DMEM medium. After culturing for 24 hours, add 20 μL of the WST-1 compound and react in the dark at 37 °C for four hours. Then, use an ELISA reader to read the absorbance value at a wavelength of 450 nm and compare it with the control group taken as 100%.

[0056] To further explore whether alkaloid compounds such as fangchinoline, cepharanoline, and cycleanine have cytotoxicity to ACE2-293T cells, the present invention performed a cytotoxicity analysis at a dose of 10 μM. The results showed that neither fangchinoline, cepharanoline, nor cycleanine caused any harm to ACE2-293T cells (Table 5 and Figure 3 ), and this result further shows that fangchinoline, cepharanoline, and cycleanine can indeed broadly inhibit virus infections mediated by the spike proteins of different SARS-CoV-2 variants.

[0057] Table 5

[0058]

[0059]

[0060] Data are expressed as mean ± standard error (n = 3)

[0061] Compared with the vehicle group (DMSO)

[0062] The present invention confirmed that Stephania sasakii and Stephania tetrandra in the herbs of the Menispermaceae family have the ability to broadly inhibit virus infections mediated by the spike proteins of different SARS-CoV-2 variants.

Claims

1. Use of a herbal extract for preparing a pharmaceutical composition for inhibiting an individual from being infected with the novel coronavirus, characterized in that, the herbal extract is an alcohol extract of a Menispermaceae herb, and the Menispermaceae herb extract refers to cepharanoline or cycleanine.

2. The use according to claim 1, characterized in that, the Menispermaceae herb refers to Stephania sasakii or Stephania tetrandra.

3. The use according to claim 1, characterized in that, the alcohol extract of the herb inhibits the novel coronavirus from infecting the individual by blocking the binding of the spike protein on the novel coronavirus to angiotensin-converting enzyme 2 on the individual's cells.

4. The use according to claim 1, characterized in that, the novel coronavirus refers to the D614G variant, the Delta variant or the Omicron variant.

5. The use according to claim 1, characterized in that, the novel coronavirus refers to the Delta variant or the Omicron variant.

6. The use according to claim 1, characterized in that, the individual is a mammal.

7. The use according to claim 1, characterized in that, the individual is a human.

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