Compound and compound for inhibiting novel coronavirus

Abstract

To provide a citrus extract, a compound, and a use for inhibiting novel coronavirus.SOLUTION: A citrus extract is made by obtaining many kinds of extracts after extraction with ethanol, ethyl acetate, and n-butanol, and by separating many kinds of compounds from an ethyl acetate extract. All of these many kinds of extracts and these many kinds of compounds have effects of inhibiting novel coronavirus.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to extracts, compounds, and uses, and in particular to citrus extracts, compounds, and uses for inhibiting the novel coronavirus. [Background technology]

[0002] COVID-19 is a global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is a novel coronavirus, also known as the novel coronavirus. It mutated from the original strain in 2019, with the Omicron variant becoming the dominant circulating strain through 2022. Despite the lower severity of the Omicron variant, antigen screening, and the emergence of next-generation vaccines, the COVID-19 pandemic continues. Summary of the Invention [Problem to be solved by the invention]

[0003] Therefore, existing technologies really need to be improved in providing substances that are naturally derived and effectively combat the COVID-19 pneumonia virus. [Means for solving the problem]

[0004] One embodiment of the present disclosure provides a citrus extract prepared by the steps of providing citrus fruit, extracting the citrus fruit with an organic solvent to obtain a liquid portion and extracted fruit, drying the liquid portion to obtain a crude extract, partitioning the crude extract with ethyl acetate and water to obtain an ethyl acetate layer and a high-polarity layer, and drying the ethyl acetate layer to obtain the ethyl acetate extract, and partitioning the high-polarity layer with n-butanol and water to obtain an n-butanol layer and an aqueous layer, drying the n-butanol layer to obtain the n-butanol extract, and drying the aqueous layer to obtain the aqueous extract.

[0005] In some embodiments, the ethyl acetate extract comprises psoralens, bergapten, 2'-hydroxy-4,4',5',6'-tetramethoxychalcone, 2'-hydroxy-3',4,4',5',6'-pentamethoxychalcone, 2'-hydroxy-3,4,4',5',6'-pentamethoxychalcone, 2'-hydroxy-3,3',4,4',5',6'-hexamethoxychalcone, 5-hydroxy-3',4',7,8-tetramethoxyflavone, 5-hydroxy-3',4',6,7,8-pentamethoxyflavone, 3',4',5,6,7,8-hexamethoxyflavone, and 3',4',5,7,8-pentamethoxyflavanone.

[0006] In some embodiments, the step of extracting citrus fruit with an organic solvent comprises extracting citrus fruit and ethanol at a weight / volume ratio of 1:5 to 15 (w / v).

[0007] Another embodiment of the present disclosure is directed to 2'-hydroxy-3',4,4',5',6'-pentamethoxychalcone, 2'-hydroxy-3,4,4',5',6'-pentamethoxychalcone, 2'-hydroxy-3,3',4,4',5',6'-hexamethoxychalcone, 5-hydroxy-3',4',7,8-tetramethoxyflavone, 3',4',5,7,8-pentamethoxyflavanone, or pharmaceutically acceptable salts thereof. The compounds are provided including acceptable salts or esters.

[0008] Another embodiment of the present disclosure provides a composition for inhibiting the novel coronavirus, comprising a crude extract, an ethyl acetate extract, an n-butanol extract, an aqueous extract, or a combination thereof, of the above-described citrus extract.

[0009] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0010] In some embodiments, the novel coronavirus comprises an original strain, an alpha variant, a beta variant, a gamma variant, a delta variant, an omicron variant, or a combination thereof.

[0011] In some embodiments, the omicron variant comprises a BA.1 subtype variant, a BA.2 subtype variant, a BA.3 subtype variant, a BA.4 subtype variant, a BA.5 subtype variant, or a combination thereof.

[0012] Another embodiment of the present disclosure provides a use of the above-described citrus extract for preparing a medicament for inhibiting a novel coronavirus, wherein the citrus extract includes a crude extract, an ethyl acetate extract, an n-butanol extract, or an aqueous extract.

[0013] Another embodiment of the present disclosure provides a use of a compound for the preparation of a drug for inhibiting a novel coronavirus, wherein the compound includes psoralen, bergapten, 2'-hydroxy-3',4,4',5',6'-pentamethoxychalcone, 2'-hydroxy-3,4,4',5',6'-pentamethoxychalcone, 2'-hydroxy-3,3',4,4',5',6'-hexamethoxychalcone, 5-hydroxy-3',4',7,8-tetramethoxyflavone, 3',4',5,6,7,8-hexamethoxyflavone, 3',4',5,7,8-pentamethoxyflavanone, or a combination thereof.

[0014] In some embodiments, the novel coronavirus comprises an original strain, an alpha variant, a beta variant, a gamma variant, a delta variant, an omicron variant, or a combination thereof.

[0015] In some embodiments, the omicron variant comprises a BA.1 subtype variant, a BA.2 subtype variant, a BA.3 subtype variant, a BA.4 subtype variant, a BA.5 subtype variant, or a combination thereof.

[0016] In some embodiments, the drug is in capsule, tablet, powder, or liquid form.

[0017] In some embodiments, the drug inhibits the binding of the novel coronavirus to the ACE2 (Angiotensin-Converting Enzyme 2) protein.

[0018] In some embodiments, the drug inhibits the novel coronavirus, thereby treating the disease caused by the novel coronavirus.

[0019] In some embodiments, the disease is severe specific infectious pneumonia (also called COVID-19 pneumonia). [Brief explanation of the drawings]

[0020] Various aspects of the present disclosure will be better understood when read in conjunction with the accompanying drawings. It should be noted that, in accordance with industry standard practice, the various features are not drawn to scale. In fact, the size of the various features may be arbitrarily increased or decreased for clarity of discussion. To make the above and other objects, features, advantages, and embodiments of the present disclosure more easily comprehensible, the following description of the accompanying drawings is provided: [Figure 1] FIG. 1 is a flow diagram illustrating a method for preparing a Citrus depressa extract according to some embodiments of the present disclosure. [Figure 2] 1 is a chromatogram of high performance liquid chromatography (HPLC) of a crude extract according to some embodiments of the present disclosure. [Figure 3] 1 is a high performance liquid chromatography chromatogram of an ethyl acetate layer extract according to some embodiments of the present disclosure. [Figure 4] 1 is a high performance liquid chromatography chromatogram of an n-butanol layer extract according to some embodiments of the present disclosure. [Figure 5] 1 is a high performance liquid chromatography chromatogram of an aqueous layer extract according to some embodiments of the present disclosure. [Figure 6]1 is a liquid chromatography-mass spectrometry (LC-MS) LC chromatogram of a crude extract according to some embodiments of the present disclosure. [Figure 7] 1 is an MS chromatogram of combined liquid chromatography-mass spectrometry of crude extracts according to some embodiments of the present disclosure. [Figure 8] 1 is a chromatogram of LC coupled liquid chromatography-mass spectrometry of an ethyl acetate layer extract according to some embodiments of the present disclosure. [Figure 9] 1 is an MS chromatogram from combined liquid chromatography-mass spectrometry of an ethyl acetate layer extract according to some embodiments of the present disclosure. [Figure 10] 1 is a chromatogram of LC coupled liquid chromatography-mass spectrometry of n-butanol layer extract according to some embodiments of the present disclosure. [Figure 11] 1 is an MS chromatogram from combined liquid chromatography-mass spectrometry of an n-butanol layer extract according to some embodiments of the present disclosure. [Figure 12] 1 is a chromatogram of LC coupled liquid chromatography-mass spectrometry of an aqueous layer extract according to some embodiments of the present disclosure. [Figure 13] 1 is an MS chromatogram from combined liquid chromatography-mass spectrometry of an aqueous layer extract according to some embodiments of the present disclosure. [Figure 14] 1 is a bar graph showing the binding rate of the novel coronavirus origin strain to ACE2 by four types of extracts according to some embodiments of the present disclosure. [Figure 15] 1 is a bar graph showing the binding rate of the novel coronavirus A1pha mutant strain to ACE2 by crude extracts according to some embodiments of the present disclosure. [Figure 16] A bar graph showing the binding rate of the novel coronavirus A1pha mutant strain to ACE2 using an ethyl acetate layer extract according to some embodiments of the present disclosure. [Figure 17]A bar graph showing the binding rate of the novel coronavirus A1pha mutant strain to ACE2 by n-butanol layer extracts according to some embodiments of the present disclosure. [Figure 18] A bar graph showing the binding rate of the novel coronavirus A1pha mutant strain to ACE2 using an aqueous layer extract according to some embodiments of the present disclosure. [Figure 19] 1 is a bar graph showing the binding rate of the novel coronavirus Beta variant to ACE2 by crude extracts according to some embodiments of the present disclosure. [Figure 20] A bar graph showing the binding rate of the novel coronavirus Beta variant to ACE2 using an ethyl acetate layer extract according to some embodiments of the present disclosure. [Figure 21] A bar graph showing the binding rate of the novel coronavirus Beta variant to ACE2 using an n-butanol layer extract according to some embodiments of the present disclosure. [Figure 22] A bar graph showing the binding rate of the novel coronavirus Beta variant to ACE2 using aqueous layer extracts according to some embodiments of the present disclosure. [Figure 23] 1 is a bar graph showing the binding rate of the novel coronavirus Delta variant to ACE2 by crude extracts according to some embodiments of the present disclosure. [Figure 24] 1 is a bar graph showing the binding rate of the novel coronavirus Delta variant to ACE2 using an ethyl acetate layer extract according to some embodiments of the present disclosure. [Figure 25] 1 is a bar graph showing the binding rate of the novel coronavirus Delta variant to ACE2 by n-butanol layer extracts according to some embodiments of the present disclosure. [Figure 26] 1 is a bar graph showing the binding rate of the novel coronavirus Delta variant to ACE2 by aqueous layer extracts according to some embodiments of the present disclosure. [Figure 27]A bar graph showing the binding rate between the RBD region (receptor-binding domain) of the novel coronavirus Delta variant and ACE2 by crude extracts according to some embodiments of the present disclosure. [Figure 28] A bar graph showing the binding rate between the RBD region and ACE2 in the novel coronavirus Delta variant using an ethyl acetate layer extract according to some embodiments of the present disclosure. [Figure 29] 1 is a bar graph showing the binding rate between the RBD region of the novel coronavirus Delta variant and ACE2 by an n-butanol layer extract according to some embodiments of the present disclosure. [Figure 30] A bar graph showing the binding rate between the RBD region of the novel coronavirus Delta variant and ACE2 using an aqueous layer extract according to some embodiments of the present disclosure. [Figure 31] 1 is a bar graph showing the binding rate of the novel coronavirus Omicron BA.1 mutant strain to ACE2 by five types of extracts according to some embodiments of the present disclosure. [Figure 32] 1 is a bar graph showing the binding rate of the novel coronavirus Omicron BA.2 mutant strain to ACE2 by four types of extracts according to some embodiments of the present disclosure. [Figure 33] 1 is a bar graph showing the binding rate of the novel coronavirus Omicron BA.4 / 5 mutant strain to ACE2 by four types of extracts according to some embodiments of the present disclosure. [Figure 34] 1 shows the binding rate of the novel coronavirus Omicron BA.2 mutant strain to ACE2 by crude extracts according to some embodiments of the present disclosure. [Figure 35] 1 shows the binding rate of the novel coronavirus Omicron BA.4 / 5 mutant strain to ACE2 by crude extracts according to some embodiments of the present disclosure. [Figure 36] 1 shows the binding rate of the novel coronavirus Omicron BA.2 mutant strain to ACE2 using an ethyl acetate layer extract according to some embodiments of the present disclosure. [Figure 37]1 shows the binding rate of the novel coronavirus Omicron BA.4 / 5 mutant strain to ACE2 using an ethyl acetate layer extract according to some embodiments of the present disclosure. [Figure 38A] 1 shows the binding rate of compound Cd1 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.1 mutant strain and ACE2. [Figure 38B] 1 shows the binding rate of compound Cd2 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.1 mutant strain and ACE2. [Figure 38C] 1 shows the binding rate of compound Cd3 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.1 mutant strain and ACE2. [Figure 38D] 1 shows the binding rate of compound Cd4 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.1 mutant strain and ACE2. [Figure 38E] 1 shows the binding rate of compound Cd5 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.1 mutant strain and ACE2. [Figure 38F] 1 shows the binding rate of compound Cd6 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.1 mutant strain and ACE2. [Figure 38G] 1 shows the binding rate of compound Cd7 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.1 mutant strain and ACE2. [Figure 38H] 1 shows the binding rate of compound Cd8 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.1 mutant strain and ACE2. [Figure 38I] 1 shows the binding rate of compound Cd9 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.1 mutant strain and ACE2. [Figure 38J] 1 shows the binding rate of compound Cd10 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.1 mutant strain and ACE2. [Figure 39A]1 shows the binding rate of compound Cd1 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.2 mutant strain and ACE2. [Figure 39B] 1 shows the binding rate of compound Cd2 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.2 mutant strain and ACE2. [Figure 39C] 1 shows the binding rate of compound Cd3 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.2 mutant strain and ACE2. [Figure 39D] 1 shows the binding rate of compound Cd4 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.2 mutant strain and ACE2. [Figure 39E] 1 shows the binding rate of compound Cd5 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.2 mutant strain and ACE2. [Figure 39F] 1 shows the binding rate of compound Cd6 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.2 mutant strain and ACE2. [Figure 39G] 1 shows the binding rate of compound Cd7 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.2 mutant strain and ACE2. [Figure 39H] 1 shows the binding rate of compound Cd8 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.2 mutant strain and ACE2. [Figure 39I] 1 shows the binding rate of compound Cd9 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.2 mutant strain and ACE2. [Figure 39J] 1 shows the binding rate of compound Cd10 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.2 mutant strain and ACE2. [Figure 40A] 1 shows the binding rate of compound Cd1 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.4 mutant strain and ACE2. [Figure 40B]1 shows the binding rate of compound Cd2 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.4 mutant strain and ACE2. [Figure 40C] 1 shows the binding rate of compound Cd3 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.4 mutant strain and ACE2. [Figure 40D] 1 shows the binding rate of compound Cd4 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.4 mutant strain and ACE2. [Figure 40E] 1 shows the binding rate of compound Cd5 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.4 mutant strain and ACE2. [Figure 40F] 1 shows the binding rate of compound Cd6 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.4 mutant strain and ACE2. [Figure 40G] 1 shows the binding rate of compound Cd7 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.4 mutant strain and ACE2. [Figure 40H] 1 shows the binding rate of compound Cd8 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.4 mutant strain and ACE2. [Figure 40I] 1 shows the binding rate of compound Cd9 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.4 mutant strain and ACE2. [Figure 40J] 1 shows the binding rate of compound Cd10 according to some embodiments of the present disclosure to the novel coronavirus Omicron BA.4 mutant strain and ACE2. DETAILED DESCRIPTION OF THE INVENTION

[0021] To provide a more detailed and complete description of the present disclosure, the following provides illustrative descriptions of the embodiments and specific examples of the present disclosure, but these are not the only ways to implement or operate the specific examples of the present disclosure. The examples disclosed below may be combined or substituted with each other when beneficial, or may be added to one example without further description or explanation. In the following description, numerous specific details are set forth in order to allow the reader to fully understand the following examples. However, the embodiments of the present disclosure may be practiced without these specific details.

[0022] In this context, unless otherwise specifically limited to the article in the text, "a," "an," and "the" may refer collectively to the singular or the plural. It should be further understood that, as used herein, the terms "comprise," "include," "have," and similar terms will identify the stated features, regions, integers, steps, operations, elements, and / or components, but will not exclude the stated or another one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof.

[0023] In this text, the term "citrus" refers to the genus Citrus (scientific name: Citrus) in the family Rutaceae, under which species include both trees and shrubs. Several important fruits belong to this genus, such as citron (Citrus medica), pomelo (Citrus maxima), ponkan (Citrus reticulata), orange (Citrus sinensis), lemon (Citrus limon), hibiscus (Citrus limonia), lime (Citrus aurantifolia), turnip (Citrus aurantium), yuzu (Citrus junos), kumquat (Citrus japonica; Cj), grapefruit (Citrus paradisi), red river orange (Citrus hongheensis), ichan papeda (Citrus ichangensis), Citrus macroptera (Citrus macroptera var. kerrii), kaffir lime (Citrus hystrix), and tachibana (Citrus tachibana), Shikuwasa (Citrus depressa; Cd), etc.

[0024] Shikuwasa, also known as Hirami Lemon, is one of the native citrus fruits of Taiwan.

[0025] In this text, the term "fruit" refers to the fruit structure, which is usually divided into two parts: the seed and the skin. The skin is further divided into the exocarp, mesocarp, and endocarp. The exocarp is soft and thick, like leather. The endocarp is the thick, juicy, fleshy part, known as the pulp. The mesocarp is the tissue between the exocarp and endocarp, and is a mostly white, firm structure compared to the endocarp.

[0026] In this paper, furanocoumarins are a series of compounds in which a hydroxy group at the 7th position of the parent nucleus is fused with a substituted isopentenyl group at the 6th or 8th position to form a furan ring (the numbering of the positions is shown, for example, in formula (I)). Psoralen belongs to the linear furanocoumarins, and bergapten is classified as an angular furanocoumarin.

[0027] In some embodiments, the organic solvent includes, but is not limited to, polar and non-polar organic solvents. Exemplary polar organic solvents include, but are not limited to, alcohols, ketones (acetone, dialkyl ketones, pyrrolidones), alkylene carbonates, alkyl esters, and aryl esters. Nonionic or anionic surfactants include, but are not limited to, carboxylates, sulfonates, natural oils, alkylamides, arylamides, alkylphenols, arylphenols, ethoxy alcohols, polyoxyethylene, carboxylic acid esters, polyalkylglycol esters, sorbitol anhydride, diol esters, carboxylic acid amides, monoalkanolamines, polyoxyethylene fatty acid amides, polysorbates, cyclodextrins, glycosyl-based (sugar-based) alcohols, silicone-based alcohols, polyalkylated alcohols, and alkylaryl ethoxides.

[0028] In this context, the term "partition" refers to the formation of layered liquids after mixing the solvent and the extractant. For example, after mixing ethyl acetate and water (1:1 by volume) with the extractant, a low polarity layer and a high polarity layer of liquid appear.

[0029] In this text, the term "novel coronavirus" refers to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as the novel coronavirus or 2019 novel coronavirus.

[0030] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of the free base or free acid and is usually biologically or otherwise undesirable.

[0031] As used herein, the term "pharmaceutically acceptable esters" refers to compounds of Formula (I) through Formula (X) that can be derivatized at functional groups to provide derivatives that are capable of conversion to the parent compound in vivo.

[0032] In some embodiments, the pharmaceutically acceptable carrier is selected from the group consisting of water, alcohols, glycols, preserving agents, antioxidants, solvents, emulsifiers, suspending agents, decomposers, binding agents, excipients, stabilizing agents, chelating agents, diluents, gelling agents, preservatives, lubricants, absorption enhancers, active agents, humectants, odor absorbers, fragrances, pH adjusting agents, occlusive agents, emollients, thickeners, solubilizing agents, penetration enhancers, and the like. The carriers include, but are not limited to, enhancers, anti-irritants, colorants, propellants, surfactants, and other carriers similar to or suitable for use in the present invention.

[0033] Spike protein binding assay

[0034] In some embodiments, an enzyme-linked immunosorbent assay (ELISA) was used to evaluate the inhibitory effect of the four extracts on the binding of SARS-COV-2 spike protein wild-type (WT, Wuhan strain) or mutant strains (α, β, γ, δ, or ο) to biotinylated human ACE2 recombinant protein. First, 100 μL of spike protein was applied to each well of a 96-well plate, diluted with buffer, and the buffer was left overnight at 4°C. The coated plate was then washed three times with a washing buffer of PBS containing Tween-20, followed by blocking with 250 μL of a blocking buffer containing bovine serum albumin (BSA) at 37°C for 1.5 hours. The 96-well plate was then washed three times, and 100 μL of the test extract or inhibitor (10 μg / mL, cat. GTX635791, GeneTex®) in dilution buffer was added to the 96-well plate and incubated at 37°C for 1 hour. Next, 100 μL of biotinylated human ACE2 protein (10 ng / mL, cat. AC2-H82E6-25ug, ACRO Biosystems®) was added to each well and incubated at 37°C for another 1 hour. Next, the plate was washed three times with 96-well plate washing buffer, and 100 μL of streptavidin-HRP coupling in dilution buffer was added and incubated at 37°C for 1 hour. The 96-well plate was then washed and incubated with 200 μL of TMB (3,3',5,5'-tetramethylbenzidine) per well for 20 minutes at 37°C, protected from light. Then, 50 μL of stop solution is added to stop the reaction, and the absorbance at 450 nm is detected using a microplate detector.

[0035] cell culture

[0036] In some embodiments, human embryonic kidney (HEK-293T / 17, ATCC® CRL-11268™) cells are obtained from the American Type Culture Collection (ATCC). Cells are cultured at 37°C in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum and 1x penicillin-streptomycin. HEK-293T-ACE2 cells are generated by transduction of a VSV-G pseudotyped lentivirus carrying the human ACE2 gene.

[0037] SARS-CoV-2 spike expression plasmid

[0038] In some embodiments, synthetic DNA fragments encoding the SARS-CoV-2 spike gene are purchased from Integrated DNA Technologies (IDT) and cloned into mammalian expression vehicles. The mutation sites for each spike mutant are shown below.

[0039] Alpha variants (B.1.1.7): 69-70 del, Y144 del, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H.

[0040] Beta mutant strain (B.1.351(501Y.V2)): L18F, D80A, D215G, 242-244 del, R246I, K417N, E484K, N501Y, D614G, A701V.

[0041] Gamma mutant strains (Lineage P1): L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I.

[0042] Delta variant (B.1.617.2): T19R, G142D, 156-157 del, R158G, L452R, T478K, D614G, P681R, D950N.

[0043] Omicron mutants (B.1.1.529): A67V, Δ69-70, T95I, G142D / Δ143-145, Δ211 / L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F.

[0044] Omicron subtype mutants BA1: A67V, Δ69-70, T95I, G142D / Δ143-145, Δ211 / L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F.

[0045] Omicron subtype mutant BA2: T19I, L24S, Δ25-27, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K.

[0046] Omicron subtype mutants BA4 / 5: T19I, L24S, Δ25-27, Δ69-70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K.

[0047] Production and purification of SARS-CoV-2 spike pseudotyped lentivirus

[0048] In some embodiments, pseudotyped lentiviruses carrying the SARS-CoV-2 spike protein are generated using pCMV-R8.91, pLAS2w.Fluc.Ppuro, and pcDNA3.1-nCoV-S (B.1.1.7, B.1.351, P1, B.1.617.2, or B.1.1.529). HEK-293T cells are inoculated the day before transfection and the indicated plasmids are delivered into the cells using TransIT-LT1 transfection reagent. The medium is changed at 16 hours and harvested at 48 and 72 hours post-transfection. Cell debris is removed by centrifugation, and the supernatant is passed through a 0.45 μm injection filter. The pseudotyped lentiviruses are aliquoted and stored at -80°C.

[0049] Assessment of lentiviral titer by cell viability assay

[0050] In some embodiments, the transduction units (TU) of SARS-CoV-2 pseudotyped lentiviruses are assessed by cell viability assays to reflect the lentivirus dilution limit. Briefly, the day before lentivirus transduction, HEK-293T cells stably expressing the human ACE2 gene are seeded into 96-well plates. For titration of pseudotyped lentiviruses, different amounts of lentivirus are added to medium containing polybrene. The 96-well plates are then infected by rotation. After culturing the cells at 37°C, the medium containing virus and polybrene is removed and replaced with fresh complete DMEM containing purinmycin. After purinmycin treatment, the medium is removed and cell viability is detected using 10% AlarmaBlue reagent according to the manufacturer's instructions. The viability of uninfected cells (without purinmycin treatment) is 100%. The viral titer (transduction units) is determined by plotting the surviving cells against the diluted viral dose.

[0051] Pseudotyped lentivirus neutralization assay

[0052] In some embodiments, for virus neutralization tests, heat-inactivated serum was serially diluted to the desired dilution concentration, and simultaneously added with extracts or single compounds. The serum was then incubated with 1,000 TU of SARS-CoV-2 pseudotyped lentivirus in DMEM (supplemented with 1% FBS and 100 U / mL penicillin / streptomycin) for 1 hour at 37°C. Then, 10,000 HEK-293T cells stably expressing the human ACE2 gene were seeded into a 96-well plate. Sixteen hours after infection, the medium was replaced with fresh complete DMEM (supplemented with 10% FBS and 100 U / mL penicillin / streptomycin), and the cells were continuously cultured for another 48 hours before luciferase assay. For luciferase assays, the expression level of the luciferase gene was measured using the Bright-Glo™ Luciferase Measurement System. Relative light units (RLU) were detected using a Molecular Devices-SpectraMaxL. The percentage of inhibition is calculated as the ratio of the reduction in RLU in the presence of diluted serum to the RLU in the serum-free control, and the calculation formula is (RLU 対照-RLU 血清 ) / RLU コントロール is.

[0053] The citrus extract of the present disclosure and its use in treating COVID-19 pneumonia will be described in more detail below with several examples and test examples. However, these examples are for illustrative purposes only and are not intended to limit the present disclosure. The scope of protection of the present disclosure is based on the content specified in the claims below.

[0054] Example

[0055] Although the methods disclosed herein are described below as a series of operations or steps, the order in which these operations or steps are presented should not be construed as a limitation on the present disclosure. For example, some operations or steps may be performed in a different order and / or simultaneously with other steps. It is not necessary to perform all operations, steps, and / or features to achieve embodiments of the present disclosure. Furthermore, each of the operations or steps described herein may include multiple sub-steps or actions.

[0056] For the sake of clarity, features and elements that are well known in the art and are not necessary for understanding the principles described may be omitted.

[0057] Example 1 Extract Preparation

[0058] Please refer to FIG. 1, which is a flow diagram showing a method for preparing a Citrus depressa extract according to one embodiment of the present disclosure.

[0059] Whole-grain shikwasa fruit was extracted with 95% ethanol at a weight / volume ratio of 1:10 (w / v) for one week to obtain a first liquid and a first-extracted shikwasa fruit. A portion of the first liquid was removed and evaporated under vacuum to obtain a dried first crude extract. Next, the first-extracted shikwasa fruit was extracted with 95% ethanol at a weight / volume ratio of 1:10 (w / v) for another week to obtain a second liquid and a second-extracted shikwasa fruit. A portion of the second liquid was removed and evaporated under vacuum to obtain a dried second crude extract. The first and second crude extracts were combined to obtain a dried crude extract.

[0060] Next, the crude extract was partitioned three times with ethyl acetate and distilled water (1:1 ratio), yielding an ethyl acetate layer (low polarity layer) and a high polarity layer. The high polarity layer was then partitioned three more times with n-butanol and distilled water (1:1 ratio), yielding an n-butanol layer and an aqueous layer. After drying each layer, the ethyl acetate layer extract, n-butanol layer extract, and aqueous layer extract were obtained.

[0061] Next, the ethyl acetate extract was collected and loaded onto a silica gel column (SNAP Cartridge KP-Sil 340 g, Biotage®) for medium pressure liquid chromatography (MPLC) to separate the bioactive substances in the ethyl acetate extract. MPLC generated 110 layers (Fr. 1 to Fr. 110) using a mobile phase of hexane and ethyl acetate (100:0 to 0:100) and acetone.

[0062] Next, layers Fr. 44, Fr. 59, Fr. 66, and Fr. 78 containing the active ingredient were selected by semi-preparative refractive index-high performance liquid chromatography (semi-preparative RI-HPLC) using a silica gel column (Luna® silica column 5 μm, 250 × 10 mm, Phenomenex®).

[0063] Layer Fr. 44 produced two furanocoumarin compounds (Cd1, Cd2) using a mobile phase consisting of hexane and ethyl acetate (8:2).

[0064] Layer Fr. 59 produced two chalcone compounds (Cd3, Cd4) using a mobile phase consisting of hexane, ethyl acetate, and dichloromethane (7:1.5:1.5).

[0065] Sublayers SubFr. 66-1 to SubFr. 66-18 were obtained by first preparing layer Fr. 66 using a silica gel column (SNAP Cartridge KP-Sil 10 g, Biotage®) and a mobile phase of hexane and ethyl acetate (100:0-0:100). SubFr. 66-14 is a flavone compound (Cd7).

[0066] Sublayer SubFr. 66-9 produced two chalcone compounds (Cd5 and Cd6) from a mobile phase of hexane and ethyl acetate (7.7:2.3).

[0067] Sublayer SubFr. 66-12 produced one flavone compound (Cd8) from a mobile phase of hexane and ethyl acetate (6.5:3.5).

[0068] Sublayer SubFr. 66-13 produced a flavone compound (Cd10) from a mobile phase of hexane and ethyl acetate (6.3:3.7).

[0069] Layer Fr. 78 produced a flavone compound (Cd9) from a mobile phase consisting of hexane and ethyl acetate (2.5:7.5). The above separation process was repeated to obtain the amount required for activity experiments.

[0070] Example 2 Analysis of the extract

[0071] The crude extract, ethyl acetate extract, n-butanol extract, and aqueous extract from Example 1 were analyzed by high performance liquid chromatography and liquid chromatography-mass spectrometry, respectively.

[0072] The experimental results are shown in Figures 2 to 13. Figure 2 is a chromatogram of high-performance liquid chromatography of the crude extract, with absorption values ​​at 220 nm, 280 nm, 320 nm, and 400 nm. Figure 3 is a chromatogram of high-performance liquid chromatography of the ethyl acetate layer extract. Figure 4 is a chromatogram of high-performance liquid chromatography of the n-butanol layer extract. Figure 5 is a chromatogram of high-performance liquid chromatography of the aqueous layer extract. Figure 6 is an LC chromatogram of the crude extract analyzed by liquid chromatography-mass spectrometry. The upper row represents positive charge and the lower row represents negative charge. Figures 7 to 13 below all have a similar layout. Figure 7 is an MS chromatogram of the crude extract analyzed by liquid chromatography-mass spectrometry. The mass-to-charge ratio (m / z) is displayed as 100 to 1500. Figures 9, 11, and 13 below all have a similar layout. Figure 8 is an LC chromatogram obtained by combined liquid chromatography-mass spectrometry of the ethyl acetate layer extract. Figure 9 is an MS chromatogram obtained by combined liquid chromatography-mass spectrometry of the ethyl acetate layer extract. Figure 10 is an LC chromatogram obtained by combined liquid chromatography-mass spectrometry of the n-butanol layer extract. Figure 11 is an MS chromatogram obtained by combined liquid chromatography-mass spectrometry of the n-butanol layer extract. Figure 12 is an LC chromatogram obtained by combined liquid chromatography-mass spectrometry of the aqueous layer extract. Figure 13 is an MS chromatogram obtained by combined liquid chromatography-mass spectrometry of the aqueous layer extract.

[0073] Example 3 Identification of Compounds

[0074] NMR tests were carried out on the above 10 compounds.

[0075] Cd1, furo[3,2-g]chromen-7-one (psoralen). 1H:6.36(d,J=9.6Hz,1H)6.81(dd,J=2.3,1.0Hz,1H)7.46(d,J=1.1Hz,1H)7.67(s,1H)7.68(d,J=2.3Hz,1H)7.78(d,J=9.6Hz,1H). 13 C:100.14,106.60,114.93,115.67,120.04,125.10,144.27,147.13,152.30,155.67,161.23.

[0076] Cd2,4-methoxyfuro[3,2-g]chromen-7-one (Bergapten). 1 H:4.24(s,3H)6.24(d,J=9.8Hz,1H)6.99(dd,J=2.4,1.0Hz,1H)7.10(t,J=1.0Hz,1H)7.57(d,J=2.4Hz,1H)8.12(dd,J=9.8,1.0Hz,1H). 13 C:60.31,94.06,105.24,106.64,112.76,112.91,139.46,145.00,149.79,152.93,158.60,161.44.

[0077] Cd 3,2'-hydroxy-4,4',5',6'-tetramethoxychalcone (E) 2'-hydroxy-4,4',5',6'-tetramethoxychalcone. 1 H:3.69(s,3H)3.81(s,3H)3.82(s,3H)3.83(s,3H)6.37(s,1H)7.01(d,J= 8Hz,2H)7.45(d,J=15.8Hz,1H)7.59(d,J=15.8Hz,1H)7.68(d,J=8Hz,2H). 13 C:55.47 56.13 60.76 61.64 96.52 110.37 114.05 114.70 115.31 118.90 124.67 124.75 128.30 129.44 130.47 134.76 143.56 153.30 158.03 158.15 161.44 192.61.

[0078] Cd 4,2'-Hydroxy-3',4,4',5',6'-pentamethoxychalcone (E) 2'-Hydroxy-3',4,4',5',6'-pentamethoxy chalcone. 1 H:3.73(s,3H)3.75(s,3H)3.77(s,3H)3.81(s,3H)3.93(s,3H)7.00(d,J=8. 8Hz,2H)7.14(d,J=16.0Hz,1H)7.42(d,J=16.0Hz,1H)7.69(d,J=8.8Hz,2H). 13 C:55.99 60.81 60.92 61.03 61.53 114.51 116.23 125.50 126.91 130.47 137.13 138.50 144.21 146.31 147.28 149.23 161.37 192.73.

[0079] Cd 5,2'-Hydroxy-3,4,4',5',6'-pentamethoxychalcone (E) 2'-Hydroxy-3,4,4',5',6'-pentamethoxychalcone. 1 H:3.81(s,3H)3.87(s,3H)3.90(s,3H)3.90(s,3H)3.91(s,3H)6.26(s,1H)6.87(d,J=8.3Hz,1H) 7.13(d,J=2.0Hz,1H)7.21(dd,J=8.3,2.0Hz,1H)7.77(d,J=15.6Hz,1H)7.81(d,J=15.6Hz,1H). 13 C:56.11 56.17 56.25 61.47 62.10 96.82 108.92 110.56 111.40 123.12 124.46 128.58 135.46 143.79 149.43 151.50 155.08 160.16 162.82 192.89.

[0080] Cd 6,2'-hydroxy-3,3',4,4',5',6'-hexamethoxychalcone (E) 2'-hydroxy-3,3',4,4',5',6'-hexamethoxy-chalcone. 1H:3.84(s,3H)3.87(s,6H)3.91(s,3H)3.92(s,3H)4.07(s,3H)6.88(d,J=8.3Hz,1H)7.13( d,J=2.0Hz,1H)7.23(dd,J=8.3,2.0Hz,1H)7.77(d,J=15.5Hz,1H)7.81(d,J=15.5Hz,1H). 13 C:56.14 56.20 61.23 61.52 61.80 62.39 110.60 111.31 111.41 123.33 128.41 137.49 138.62 144.38 149.47 151.02 151.68 153.51 155.12 193.69.

[0081] Cd 7,5-Hydroxy-3',4',7,8-tetramethoxyflavone 5-Hydroxy-3',4',7,8-tetramethoxyflavone 1 H:3.92(s,3H)3.93(s,6H)3.95(s,3H)3.96(s,3H)6.41(s,1H)6.57(s,1H)6.98(d,J=8.5Hz,1H)7.41(d,J=2.1Hz,1H)7.57(dd,J=8.5,2.1Hz,1H). 13 C:56.22 56.34 56.56 61.79 95.98 104.28 105.03 109.06 111.50 120.38 124.04 129.17 149.58 149.64 152.63 157.79 158.82 164.06 182.85.

[0082] Cd8,5-hydroxy-3',4',6,7,8-pentamethoxyflavone 1 H:3.93(s,3H)3.94(s,3H)3.95(s,6H)4.09(s,3H)6.59(s,1H)6.97(d,J=8.4Hz,1H)7.40(d,J=2.1Hz,1H)7.56(dt,J=8.4,2.1Hz,1H). 13C:56.20 56.32 61.32 61.91 62.25 104.16 107.17 109.00 111.49 120.37 123.88 133.15 136.78 145.97 149.59 149.72 152.69 153.20 164.15 183.18.

[0083] Cd9,3',4',5,6,7,8-hexamethoxyflavone3',4',5,6,7,8-hexamethoxyflavone (Nobiletin). 1 H:3.86(s,9H)3.88(s,3H)3.94(s,3H)4.02(s,3H)6.52(s,1H)6.90(d,J=8.5Hz,1H)7.32(d,J=2.1Hz,1H)7.47(dd,J=8.5,2.1Hz,1H). 13 C:55.99 56.09 61.66 61.81 61.96 62.25 106.82 108.63 111.31 114.83 119.67 123.99 138.04 144.10 147.73 148.39 149.32 151.45 151.99 161.10 177.36 206.96.

[0084] Cd10,3',4',5,7,8-pentamethoxyflavanone 3',4',5,7,8 -pentamethoxyflavanone 1 H: 2.70(dd,J=16.6,2.8Hz,1H)2.97(dd,J=16.6,13.4Hz,1H)3.75(s,3H)3.81(s,3H)3.83(s,3H)3.85(s,3H)3.87(s,3 H)5.28(dd,J=13.4,2.8Hz,1H)6.29(s,1H)6.84(d,J=,8.0Hz,1H)6.92(dd,J=2.0,8.0Hz,1H)6.93(d,J=2.0Hz,1H). Table 1: Chemical structures of 10 compounds JPEG2025186512000002.jpg139114

[0085] Example 4: Inhibitory effect of the extract on the binding of the novel coronavirus to ACE2

[0086] An enzyme-linked immunosorbent assay (ELISA) was used to evaluate the inhibitory effects of four extracts on the binding of SARS-CoV-2 spike protein wild-type (WT, Wuhan strain) or mutants (α, β, δ, or o) to biotinylated human ACE2 recombinant protein. Experiments were primarily divided into a control group (Blank; B), a control group (also known as the ACE2 group, i.e., ACE2 and virus alone, without extract), an inhibitor group (Inhibitor, Inh., 10 μg / mL, cat. GTX635791, GeneTex®, recognizing the full-length SARS-CoV-2 RBD recombinant protein (Wuhan-Hu-1 strain)), and various extract groups (e.g., crude extract, ethyl acetate extract, n-butanol extract, and aqueous extract).

[0087] Please refer to Figure 14, which is a bar graph showing the binding rate of the novel coronavirus origin strain to ACE2 for the four types of extracts (5 mg / mL) in Example 1. As can be seen from the results, the crude extract, ethyl acetate layer extract, n-butanol layer extract, and aqueous layer extract group all significantly inhibited the binding of the novel coronavirus origin strain to ACE2.

[0088] Please refer to Figure 15, which is a bar graph showing the binding rate between the novel coronavirus A1pha mutant strain and ACE2 using the crude extract of Example 1. As can be seen from the results, crude extracts at concentrations of 0.5 mg / mL to 2 mg / mL significantly inhibited the binding between the novel coronavirus A1pha mutant strain and ACE2.

[0089] Please refer to Figure 16, which is a bar graph showing the binding rate between the novel coronavirus A1pha mutant strain and ACE2 using the ethyl acetate layer extract of Example 1. As can be seen from the results, the ethyl acetate layer extract at concentrations of 0.25 mg / mL to 2 mg / mL significantly inhibited the binding between the novel coronavirus A1pha mutant strain and ACE2.

[0090] Please refer to Figure 17, which is a bar graph showing the binding rate of the novel coronavirus A1pha mutant strain to ACE2 using the n-butanol layer extract of Example 1. As can be seen from the results, the n-butanol layer extract at a concentration of 2 mg / mL tended to inhibit the binding of the novel coronavirus A1pha mutant strain to ACE2.

[0091] Please refer to Figure 18, which is a bar graph showing the binding rate of the novel coronavirus A1pha mutant strain to ACE2 by the aqueous extract of Example 1. As can be seen from the results, the aqueous extract at each concentration did not significantly inhibit the binding of the novel coronavirus A1pha mutant strain to ACE2.

[0092] Please refer to Figure 19, which is a bar graph showing the binding rate between the novel coronavirus Beta variant and ACE2 using the crude extract of Example 1. As can be seen from the results, the crude extract at concentrations of 0.5 mg / mL and 2 mg / mL significantly inhibited the binding between the novel coronavirus Beta variant and ACE2.

[0093] Please refer to Figure 20, which is a bar graph showing the binding rate between the novel coronavirus Beta mutant strain and ACE2 using the ethyl acetate layer extract of Example 1. As can be seen from the results, the ethyl acetate layer extract at concentrations of 0.25 mg / mL to 2 mg / mL significantly inhibited the binding between the novel coronavirus Beta mutant strain and ACE2.

[0094] Please refer to Figure 21, which is a bar graph showing the binding rate between the novel coronavirus Beta mutant strain and ACE2 using the n-butanol layer extract of Example 1. As can be seen from the results, the n-butanol layer extract at a concentration of 2 mg / mL significantly inhibited the binding between the novel coronavirus Beta mutant strain and ACE2, and the n-butanol layer extract at concentrations of 0.25 mg / mL to 0.5 mg / mL also tended to slightly inhibit the binding.

[0095] Please refer to Figure 22, which is a bar graph showing the binding rate between the novel coronavirus Beta mutant strain and ACE2 using the aqueous layer extract of Example 1. As can be seen from the results, aqueous layer extracts with concentrations of 0.5 mg / mL to 2 mg / mL significantly inhibited the binding between the novel coronavirus Beta mutant strain and ACE2.

[0096] Please refer to Figure 23, which is a bar graph showing the binding rate between the novel coronavirus Delta variant and ACE2 using the crude extract of Example 1. As can be seen from the results, crude extracts at concentrations of 1.25 mg / mL to 10 mg / mL significantly inhibited the binding between the novel coronavirus Delta variant and ACE2.

[0097] Please refer to Figure 24, which is a bar graph showing the binding rate between the novel coronavirus Delta variant and ACE2 using the ethyl acetate extract of Example 1. As can be seen from the results, the ethyl acetate extract at concentrations of 1.25 mg / mL to 10 mg / mL significantly inhibited the binding between the novel coronavirus Delta variant and ACE2.

[0098] Please refer to Figure 25, which is a bar graph showing the binding rate of the novel coronavirus Delta variant to ACE2 using the n-butanol layer extract of Example 1. As can be seen from the results, n-butanol layer extracts with concentrations of 1.25 mg / mL to 2.5 mg / mL tend to inhibit the binding of the novel coronavirus Delta variant to ACE2.

[0099] Please refer to Figure 26, which is a bar graph showing the binding rate of the novel coronavirus Delta variant to ACE2 by the aqueous layer extract of Example 1. As can be seen from the results, the aqueous layer extract did not significantly inhibit the binding of the novel coronavirus Delta variant to ACE2.

[0100] Please refer to Figure 27, which is a bar graph showing the binding rate between the RBD region and ACE2 in the novel coronavirus Delta mutant strain using the crude extract of Example 1. As can be seen from the results, crude extracts at concentrations of 1.25 mg / mL to 10 mg / mL significantly inhibited the binding between the RBD region and ACE2 in the novel coronavirus Delta mutant strain.

[0101] Please refer to Figure 28, which is a bar graph showing the binding rate between the RBD region and ACE2 in the novel coronavirus Delta mutant strain using the ethyl acetate layer extract of Example 1. As can be seen from the results, the ethyl acetate layer extract at concentrations of 1.25 mg / mL to 10 mg / mL significantly inhibited the binding between the RBD region and ACE2 in the novel coronavirus Delta mutant strain.

[0102] Please refer to Figure 29, which is a bar graph showing the binding rate between the RBD region and ACE2 in the novel coronavirus Delta mutant strain using the n-butanol layer extract of Example 1. As can be seen from the results, n-butanol layer extract at concentrations of 1.25 mg / mL to 5 mg / mL tended to or significantly inhibited the binding between the RBD region and ACE2 in the novel coronavirus Delta mutant strain.

[0103] Please refer to Figure 30, which is a bar graph showing the binding rate between the RBD region and ACE2 in the novel coronavirus Delta variant by the aqueous extract of Example 1. As can be seen from the results, the aqueous extract at each concentration did not significantly inhibit the binding between the RBD region and ACE2 in the novel coronavirus Delta variant.

[0104] Please refer to Figure 31, which is a bar graph showing the binding rate of the novel coronavirus Omicron BA.1 variant to ACE2 using the four extracts from Example 1. As can be seen from the results, the 2 mg / mL crude extract, ethyl acetate extract, n-butanol extract, and aqueous extract all significantly inhibited the binding of the novel coronavirus Omicron BA.1 variant to ACE2. The preparation method for the 2 mg / mL kumquat crude extract (Cj) was similar to that of Example 1 (extraction with alcohol), and it also significantly inhibited the binding of the novel coronavirus Omicron BA.1 variant to ACE2. Furthermore, Layer Fr.82 promoted the binding of the novel coronavirus Omicron BA.1 variant to ACE2.

[0105] Please refer to Figure 32, which is a bar graph showing the binding rate between the novel coronavirus Omicron BA.2 mutant strain and ACE2 using the four extracts in Example 1. As can be seen from the results, the 2 mg / mL crude extract, ethyl acetate extract, n-butanol extract, and aqueous extract all significantly inhibited the binding between the novel coronavirus Omicron BA.2 mutant strain and ACE2, and the aqueous extract also tended to inhibit binding.

[0106] Please refer to Figure 33, which is a bar graph showing the binding rate of the novel coronavirus Omicron BA.4 / 5 mutant strain to ACE2 by four types of extracts according to some embodiments of the present disclosure (the sequences of the BA.4 and BA.5 binding sites are the same). As can be seen from the results, the 2 mg / mL crude extract, ethyl acetate extract, n-butanol extract, and aqueous extract all significantly inhibited the binding of the novel coronavirus Omicron BA.4 / 5 mutant strain to ACE2. Also, as can be seen from the figure, the inhibitors did not effectively inhibit the binding of the novel coronavirus Omicron BA.4 / 5 mutant strain to ACE2.

[0107] Example 5: Neutralization test of the novel coronavirus by extracts and compounds

[0108] For the virus neutralization test, 1,000 TU of SARS-CoV-2 pseudotyped lentivirus was used to inoculate HEK-293T cells stably expressing 10,000 human ACE2 genes in a 96-well plate. At the same time, different concentrations of the extracts or compounds in Example 1 were added, and the binding of the virus to ACE2 was observed, and the half-maximal inhibitory concentration (IC) was calculated. 50 was recorded.

[0109] Please refer to Figure 34, which shows the binding rate of the novel coronavirus Omicron BA.2 mutant strain to ACE2 by the crude extract of Example 1. As can be seen from the results, the half maximal inhibitory concentration (IC 50 was 77.8 μg / mL.

[0110] Please refer to Figure 35, which shows the binding rate of the crude extract of Example 1 between the novel coronavirus Omicron BA.4 / 5 mutant strain and ACE2. As can be seen from the results, the half maximal inhibitory concentration (IC 50 was 77.4 μg / mL.

[0111] Please refer to Figure 36, which shows the binding rate of the novel coronavirus Omicron BA.2 mutant strain to ACE2 by the ethyl acetate extract of Example 1. As can be seen from the results, the half maximal inhibitory concentration (IC 50 was 74 μg / mL.

[0112] Please refer to Figure 37, which shows the binding rate of the novel coronavirus Omicron BA.4 / 5 mutant strain to ACE2 by the ethyl acetate layer extract of Example 1. As can be seen from the results, the half maximal inhibitory concentration (IC 50 was 100 μg / mL.

[0113] Please refer to Figures 38A to 38J, which show the binding rates of compounds Cd1 to Cd10 of Example 1 to the novel coronavirus Omicron BA.1 mutant strain and ACE2. As can be seen from the results, the half-maximal inhibitory concentrations IC 50were 355.63 μM, 248.31 μM, 70.47 μM, N / A, 124.17 μM, 240.44 μM, 56.75 μM, 69.98 μM, 403.65 μM, and 180.72 μM, respectively.

[0114] Please refer to Figures 39A to 39J, which show the binding rates of compounds Cd1 to Cd10 of Example 1 to the novel coronavirus Omicron BA.2 mutant strain and ACE2. As can be seen from the results, the half-maximal inhibitory concentrations IC 50 were 307.61 μM, 219.28 μM, 74.82 μM, 181.97 μM, 52.36 μM, 134.28 μM, 80.17 μM, 79.25 μM, 69.50 μM, and 114.29 μM, respectively.

[0115] Please refer to Figures 40A to 40J, which show the binding rates of compounds Cd1 to Cd10 of Example 1 to the novel coronavirus Omicron BA.4 / 5 mutant strain and ACE2. As can be seen from the results, the half inhibitory concentrations IC 50 were 161.06 μM, 205.59 μM, 64.57 μM, 125.60 μM, 70.31 μM, 175.79 μM, 104.23 μM, 16.22 μM, 39.36 μM, and 87.10 μM, respectively.

[0116] In some embodiments, the crude extract, the ethyl acetate layer extract, the n-butanol layer extract, and the aqueous layer extract each have the effect of inhibiting the binding of the novel coronavirus origin strain to ACE2.

[0117] In some embodiments, the crude extract and the ethyl acetate extract have the effect of inhibiting the binding of the novel coronavirus A1pha mutant strain to ACE2.

[0118] In some embodiments, crude extracts, ethyl acetate layer extracts, n-butanol layer extracts at greater than 2 mg / mL, and aqueous layer extracts at greater than 0.5 mg / mL have the effect of inhibiting the binding of novel coronavirus Beta variants to ACE2.

[0119] In some embodiments, the crude extract, the ethyl acetate layer extract, and the n-butanol layer extract at a concentration of 1.25 to 2.5 mg / mL have the effect of inhibiting the binding of the novel coronavirus Delta variant to ACE2.

[0120] In some embodiments, the crude extract, ethyl acetate layer extract, and n-butanol layer extract at a concentration of 1.25 to 5 mg / mL have the effect of inhibiting the binding of the RBD region of the novel coronavirus Delta mutant strain to ACE2.

[0121] In some embodiments, the crude extract, the ethyl acetate layer extract, and the n-butanol layer extract have the effect of inhibiting the binding of the novel coronavirus BA.1 and BA.2 mutant strains to ACE2.

[0122] In some embodiments, the crude extract, the ethyl acetate layer extract, the n-butanol layer extract, and the aqueous layer extract have the effect of inhibiting the binding of the novel coronavirus BA.4 / 5 mutant strain to ACE2.

[0123] In some embodiments, compounds Cd1 to Cd10 have the effect of inhibiting the binding of novel coronavirus to ACE2.

[0124] Although the present disclosure has disclosed the embodiments as described above, this does not limit the present disclosure, and anyone skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure. Therefore, the scope of protection of the present disclosure is determined based on the content specified in the following claims.

Claims

1. A compound selected from the group consisting of 2'-hydroxy-3',4,4',5',6'-pentamethoxychalcone, 2'-hydroxy-3,4,4',5',6'-pentamethoxychalcone, 2'-hydroxy-3,3',4,4',5',6'-hexamethoxychalcone, 5-hydroxy-3',4',7,8-tetramethoxyflavone, 3',4',5,7,8-pentamethoxyflavanone, and pharmaceutically acceptable salts or esters thereof.

2. A compound for use in inhibiting a novel coronavirus, the compound comprising psoralen, bergapten, 2'-hydroxy-3',4,4',5',6'-pentamethoxychalcone, 2'-hydroxy-3,4,4',5',6'-pentamethoxychalcone, 2'-hydroxy-3,3',4,4',5',6'-hexamethoxychalcone, 5-hydroxy-3',4',7,8-tetramethoxyflavone, 3',4',5,6,7,8-hexamethoxyflavone, 3',4',5,7,8-pentamethoxyflavanone, or a combination thereof.

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