Composition for suppressing the novel coronavirus

Abstract

To provide citrus extracts, compounds, and applications for inhibiting novel coronavirus.SOLUTION: This invention provides a citrus extract which, after extraction with ethanol, ethyl acetate, and n-butanol, yields multiple extracts. Furthermore, multiple compounds are isolated from the ethyl acetate extract. These multiple extracts and compounds have the effect of inhibiting the novel coronavirus.SELECTED DRAWING: Figure 1

Description

[Technical Field]

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

[0002] Severe special infectious pneumonia (COVID-19) is a global pandemic caused by severe acute respiratory syndrome coronavirus type 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 until 2022. Despite the lower severity rates associated with the Omicron variant, antigen screening, and the emergence of next-generation vaccines, the pandemic has not yet subsided. [Overview of the project] [Problems that the invention aims to solve]

[0003] Therefore, existing technologies need to be improved in order to provide a naturally derived substance that effectively combats the pneumonia virus caused by COVID-19. [Means for solving the problem]

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

[0005] In some embodiments, the ethyl acetate extract comprises psoralen, 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 includes extracting citrus fruit and ethanol in a weight-to-volume ratio of 1:5 to 15 (w / v).

[0007] Another embodiment of the present disclosure is 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 the pharmaceutically equivalent thereof The present invention provides compounds containing acceptable salts or esters.

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

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

[0010] In some embodiments, the novel coronavirus includes the origin strain, alpha mutant strain, beta mutant strain, gamma mutant strain, delta mutant strain, omicron mutant strain, or a combination thereof.

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

[0012] Another embodiment of the present disclosure is the use for the preparation of a drug for suppressing novel coronavirus of the citrus extract described above, wherein the citrus extract includes a crude extract, an ethyl acetate extract, an n-butanol extract, or an aqueous layer extract.

[0013] Another embodiment of the present disclosure is the use for the preparation of a drug for suppressing novel coronavirus of a compound, wherein the compound includes psoralen, bergapten, 2'-hydroxy-3',4,4',5',6'-pentamethoxy chalcone, 2'-hydroxy-3,4,4',5',6'-pentamethoxy chalcone, 2'-hydroxy-3,3',4,4',5',6'-hexamethoxy chalcone, 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 includes the original strain, the alpha variant, the beta variant, the gamma variant, the delta variant, the Omicron variant, or a combination thereof.

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

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

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

[0018] In some embodiments, the drug treats a disease caused by the novel coronavirus by inhibiting the novel coronavirus.

[0019] In some embodiments, the disease is severe special infectious pneumonia (or also called novel coronavirus pneumonia).

Brief Description of the Drawings

[0020] The various aspects of the present disclosure will be better understood by reading the following detailed description in conjunction with the accompanying drawings. It should be noted that according to industry standard practices, the various feature structures are not drawn to a fixed scale. Substantially, for the sake of clearer consideration, the sizes of the various feature structures may be arbitrarily enlarged or reduced. To make the above and other objects, features, merits and examples of the present disclosure clearer, the description of the accompanying drawings is as follows. [Figure 1] It is a flowchart showing a method for preparing a seekwarser extract according to some embodiments of the present disclosure. [Figure 2] It is a chromatogram of high performance liquid chromatography (HPLC) of a crude extract according to some embodiments of the present disclosure. [Figure 3] It is a chromatogram of high performance liquid chromatography of an ethyl acetate layer extract according to some embodiments of the present disclosure. [Figure 4] It is a chromatogram of high performance liquid chromatography of an n-butanol layer extract according to some embodiments of the present disclosure. [Figure 5] It is a chromatogram of high performance liquid chromatography of an aqueous layer extract according to some embodiments of the present disclosure. [Figure 6]These are LC chromatograms obtained by liquid chromatography-mass spectrometry (LC-MS) of crude extracts according to some embodiments of this disclosure. [Figure 7] These are MS chromatograms obtained using a liquid chromatography-mass spectrometry combination method for crude extracts according to some embodiments of this disclosure. [Figure 8] These are chromatograms of LC obtained by liquid chromatography-mass spectrometry in combination with liquid chromatography-mass spectrometry of ethyl acetate layer extracts according to some embodiments of the present disclosure. [Figure 9] This is an MS chromatogram of an ethyl acetate layer extract obtained using liquid chromatography-mass spectrometry in combination with some embodiments of the present disclosure. [Figure 10] These are chromatograms of LC obtained by liquid chromatography-mass spectrometry in combination with some embodiments of the present disclosure of n-butanol layer extracts. [Figure 11] This is an MS chromatogram obtained by liquid chromatography-mass spectrometry of an n-butanol layer extract according to some embodiments of this disclosure. [Figure 12] These are chromatograms of LC in a combined liquid chromatography-mass spectrometry (LC) of aqueous layer extracts according to some embodiments of the present disclosure. [Figure 13] This is an MS chromatogram of an aqueous layer extract in a liquid chromatography-mass spectrometry combination according to some embodiments of the present disclosure. [Figure 14] This is a bar graph showing the binding rates of four types of extracts according to some embodiments of this disclosure to the novel coronavirus-derived strain and ACE2. [Figure 15] This bar graph shows the binding rate between the A1pha variant of the novel coronavirus and ACE2 using crude extracts according to some embodiments of this disclosure. [Figure 16] This bar graph shows the binding rate between the A1pha variant of the novel coronavirus and ACE2 using ethyl acetate layer extracts according to some embodiments of this disclosure. [Figure 17]This bar graph shows the binding rate between the A1pha variant of the novel coronavirus and ACE2 using n-butanol layer extracts according to some embodiments of this disclosure. [Figure 18] This bar graph shows the binding rate between the A1pha variant of the novel coronavirus and ACE2 using aqueous layer extracts according to some embodiments of this disclosure. [Figure 19] This bar graph shows the binding rate between the Beta variant of the novel coronavirus and ACE2 using crude extracts according to some embodiments of this disclosure. [Figure 20] This bar graph shows the binding rate between the ethyl acetate layer extract of the novel coronavirus Beta mutant and ACE2 according to some embodiments of this disclosure. [Figure 21] This bar graph shows the binding rate between the Beta variant of the novel coronavirus and ACE2 using n-butanol layer extract according to some embodiments of this disclosure. [Figure 22] This bar graph shows the binding rate between the Beta variant of the novel coronavirus and ACE2 using aqueous layer extracts according to some embodiments of this disclosure. [Figure 23] This bar graph shows the binding rate between the Delta variant of the novel coronavirus and ACE2 using crude extracts according to some embodiments of this disclosure. [Figure 24] This bar graph shows the binding rate between the ethyl acetate layer extract of the novel coronavirus Delta mutant and ACE2 according to some embodiments of this disclosure. [Figure 25] This bar graph shows the binding rate of n-butanol layer extracts to the SARS-CoV-2 Delta mutant and ACE2 according to some embodiments of this disclosure. [Figure 26] This bar graph shows the binding rate between the Delta variant of the novel coronavirus and ACE2 using aqueous layer extracts according to some embodiments of this disclosure. [Figure 27]This bar graph shows the binding rate between the RBD region (receptor-binding domain) and ACE2 in the Delta variant of the novel coronavirus, based on crude extracts according to some embodiments of this disclosure. [Figure 28] This bar graph shows the binding rate between the RBD region and ACE2 in the Delta variant of the novel coronavirus using ethyl acetate layer extract according to some embodiments of this disclosure. [Figure 29] This bar graph shows the binding rate between the RBD region and ACE2 in the Delta variant of the novel coronavirus using n-butanol layer extract according to some embodiments of this disclosure. [Figure 30] This bar graph shows the binding rate between the RBD region and ACE2 in the Delta variant of the novel coronavirus using aqueous layer extracts according to some embodiments of this disclosure. [Figure 31] This bar graph shows the binding rates of five different extracts according to some embodiments of this disclosure to the Omicron BA.1 mutant strain of the novel coronavirus and ACE2. [Figure 32] This bar graph shows the binding rates of four types of extracts according to some embodiments of this disclosure to the Omicron BA.2 mutant strain of the novel coronavirus and ACE2. [Figure 33] This bar graph shows the binding rates of four types of extracts according to some embodiments of this disclosure to the Omicron BA.4 / 5 mutant strain of the novel coronavirus and ACE2. [Figure 34] The binding rates of crude extracts of the novel coronavirus Omicron BA.2 mutant to ACE2 according to some embodiments of this disclosure are shown. [Figure 35] The binding rates of crude extracts according to several embodiments of this disclosure to the Omicron BA.4 / 5 mutant of the novel coronavirus and ACE2 are shown. [Figure 36] The binding rates of ethyl acetate layer extracts to the Omicron BA.2 mutant of the novel coronavirus and ACE2 according to some embodiments of this disclosure are shown. [Figure 37]The binding rates of ethyl acetate layer extracts to the Omicron BA.4 / 5 mutant of the novel coronavirus and ACE2 according to some embodiments of this disclosure are shown. [Figure 38A] The binding rates of compound Cd1 according to several embodiments of this disclosure to the Omicron BA.1 mutant of the novel coronavirus and ACE2 are shown. [Figure 38B] The binding rates of compound Cd2 according to some embodiments of this disclosure to the Omicron BA.1 mutant of the novel coronavirus are shown. [Figure 38C] The binding rates of compound Cd3 according to some embodiments of this disclosure to the Omicron BA.1 mutant of the novel coronavirus and ACE2 are shown. [Figure 38D] The binding rates of compound Cd4 according to some embodiments of this disclosure to the Omicron BA.1 mutant of the novel coronavirus and ACE2 are shown. [Figure 38E] The binding rates of compound Cd5 according to some embodiments of this disclosure to the Omicron BA.1 mutant of the novel coronavirus and ACE2 are shown. [Figure 38F] The binding rates of compound Cd6 according to some embodiments of this disclosure to the Omicron BA.1 mutant of the novel coronavirus and ACE2 are shown. [Figure 38G] The binding rates of compound Cd7 according to several embodiments of this disclosure to the Omicron BA.1 mutant of the novel coronavirus and ACE2 are shown. [Figure 38H] The binding rates of compound Cd8 according to some embodiments of this disclosure to the Omicron BA.1 mutant of the novel coronavirus and ACE2 are shown. [Figure 38I] The binding rates of compound Cd9 according to some embodiments of this disclosure to the Omicron BA.1 mutant of the novel coronavirus and ACE2 are shown. [Figure 38J] The binding rates of compound Cd10 according to some embodiments of this disclosure to the Omicron BA.1 mutant of the novel coronavirus and ACE2 are shown. [Figure 39A]The binding rates of compound Cd1 according to several embodiments of this disclosure to the Omicron BA.2 mutant of the novel coronavirus and ACE2 are shown. [Figure 39B] The binding rates of compound Cd2 according to some embodiments of this disclosure to the Omicron BA.2 mutant of the novel coronavirus and ACE2 are shown. [Figure 39C] The binding rates of compound Cd3 according to some embodiments of this disclosure to the Omicron BA.2 mutant of the novel coronavirus and ACE2 are shown. [Figure 39D] The binding rates of compound Cd4 according to some embodiments of this disclosure to the Omicron BA.2 mutant of the novel coronavirus and ACE2 are shown. [Figure 39E] The binding rates of compound Cd5 according to several embodiments of this disclosure to the Omicron BA.2 mutant of the novel coronavirus and ACE2 are shown. [Figure 39F] The binding rates of compound Cd6 according to some embodiments of this disclosure to the Omicron BA.2 mutant of the novel coronavirus and ACE2 are shown. [Figure 39G] The binding rates of compound Cd7 according to some embodiments of this disclosure to the Omicron BA.2 mutant of the novel coronavirus and ACE2 are shown. [Figure 39H] The binding rates of compound Cd8 according to several embodiments of this disclosure to the Omicron BA.2 mutant of the novel coronavirus and ACE2 are shown. [Figure 39I] The binding rates of compound Cd9 according to some embodiments of this disclosure to the Omicron BA.2 mutant of the novel coronavirus and ACE2 are shown. [Figure 39J] The binding rates of compound Cd10 according to some embodiments of this disclosure to the Omicron BA.2 mutant of the novel coronavirus and ACE2 are shown. [Figure 40A] The binding rates of compound Cd1 according to several embodiments of this disclosure to the Omicron BA.4 mutant of the novel coronavirus and ACE2 are shown. [Figure 40B]The binding rates of compound Cd2 according to several embodiments of this disclosure to the Omicron BA.4 mutant of the novel coronavirus are shown. [Figure 40C] The binding rates of compound Cd3 according to some embodiments of this disclosure to the Omicron BA.4 mutant of the novel coronavirus and ACE2 are shown. [Figure 40D] The binding rates of compound Cd4 according to several embodiments of this disclosure to the Omicron BA.4 mutant of the novel coronavirus and ACE2 are shown. [Figure 40E] The binding rates of compound Cd5 according to some embodiments of this disclosure to the Omicron BA.4 mutant of the novel coronavirus and ACE2 are shown. [Figure 40F] The binding rates of compound Cd6 according to some embodiments of this disclosure to the Omicron BA.4 mutant of the novel coronavirus and ACE2 are shown. [Figure 40G] The binding rates of compound Cd7 according to some embodiments of this disclosure to the Omicron BA.4 mutant of the novel coronavirus and ACE2 are shown. [Figure 40H] The binding rates of compound Cd8 according to some embodiments of this disclosure to the Omicron BA.4 mutant of the novel coronavirus and ACE2 are shown. [Figure 40I] The binding rates of compound Cd9 according to some embodiments of this disclosure to the Omicron BA.4 mutant of the novel coronavirus and ACE2 are shown. [Figure 40J] The binding rates of compound Cd10 according to some embodiments of this disclosure to the Omicron BA.4 mutant of the novel coronavirus and ACE2 are shown. [Modes for carrying out the invention]

[0021] To make the description of this disclosure more detailed and complete, the following descriptive descriptions of embodiments and specific examples of this disclosure are provided, but this is not the only way to implement or operate the specific examples of this disclosure. Each example disclosed below may be combined or replaced with one another as usefully as possible, and other examples may be added to one example without further description or explanation. In the following descriptions, numerous specific details are described in detail to help the reader fully understand the following examples. However, the examples of this disclosure can be put into practice even without these specific details.

[0022] In this text, unless otherwise specifically limited in the text, “one” and “the aforementioned” may refer to one or more items collectively. Furthermore, it should be understood that the terms “equipped with,” “included,” “possessing,” and similar terms used herein specify the features, regions, integers, processes, operations, elements and / or components described, but do not exclude one or more other features, regions, integers, processes, operations, elements, components and / or groups thereof that are described.

[0023] In this text, the term "citrus fruits" refers to the genus Citrus (scientific name: Citrus) belonging to the Rutaceae family, and its species include both trees and shrubs. Several important fruits belong to this genus, for example, citron (Citrus medica), pomelo (Citrus maxima), ponkan (Citrus reticulata), orange (Citrus sinensis), lemon (Citrus limon), dwarf lemon (Citrus limonia), lime (Citrus aurantifolia), cucumber (Citrus aurantium), yuzu (Citrus junos), kumquat (Citrus japonica; Cj), grapefruit (Citrus paradisi), Akagawa orange (Citrus hongheensis), ichanpapeda (Citrus ichangensis), citrus macroptera (Citrus macroptera var. kerrii), kaffir lime (Citrus hystrix), and tachibana (Citrus Examples include tachibana, shikwasa (Citrus depressa; Cd), etc.

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

[0025] In this text, the term "fruit" refers to the fact that the structure of a fruit is usually divided into two parts: the seed and the peel. The peel 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, the so-called pulp. The mesocarp is the tissue between the exocarp and the endocarp, and is mostly white and firmer than the endocarp.

[0026] In this text, furocoumarines are a series of compounds in which a hydroxyl group at position 7 of the parent molecule condenses with a substituted isopentenyl group at position 6 or 8 to form a furan ring (position numbers are shown, for example, in formula (I)). Psoralen belongs to the linear furocoumarine group, while bergapten is classified as a horn-type furocoumarine.

[0027] In some embodiments, organic solvents include, but are not limited to, polar and nonpolar organic solvents. For example, polar organic solvents include, but are not limited to, alcohols, ketones (acetone, dialkylketones, pyrrolidones), alkylene carbonates, alkyl esters, and aryl esters. Nonionic or anionic surfactants include, but are not limited to, the group consisting of carboxylates, sulfonates, natural oils, alkylamides, arylamides, alkylphenols, arylphenols, ethoxyalcohols, polyoxyethylene, carboxylic acid esters, polyalkylene glycol esters, anhydrous sorbitol, diol esters, carboxylic acid amides, monoalkanolamines, polyoxyethylene fatty acid amides, polysorbates, cyclodextrins, sugar-based alcohols, silicone-based alcohols, polyalkylated alcohols, and alkylaryl ethoxides.

[0028] In this text, the term "partition" refers to the formation of layered liquids after mixing a solvent and the substance to be extracted. For example, after mixing ethyl acetate, water (1:1 volume), and the substance to be extracted, two liquid layers appear: a low-polarity layer and a high-polarity layer.

[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 the 2019 novel coronavirus.

[0030] In this text, the term “medically acceptable salt” means a salt that retains the biological efficacy and properties of a free base or free acid and is generally biologically or otherwise ideal.

[0031] In this text, the term "pharmaceutically acceptable ester" refers to a compound of formulas (I) to (X) that can be derivatized on a functional group to provide a derivative that can be converted back to the parent compound in vivo.

[0032] In some embodiments, pharmaceutically acceptable carriers include 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, and penetration enhancers. This includes, but is not limited to, enhancers, anti-irritants, colorants, propellants, surfactants, and other carriers similar to or suitable for the present invention.

[0033] Measurement of spike protein binding

[0034] In several embodiments, experiments were performed using enzyme-linked immunosorbent assay (ELISA) to evaluate the inhibitory performance of 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 coated into each well of a 96-well plate and diluted with buffer, and the buffer was left overnight at 4°C. Then, the coated plate was washed three times with PBS washing buffer containing Tween-20, and then blocked with a blocking buffer containing 250 μL of fetal bovine serum albumin (BSA) at 37°C for 1.5 hours. Next, the 96-well plate is washed three times, and 100 μL of the test extract or inhibitor (10 μg / mL, cat.GTX635791, GeneTex®) in dilution buffer is 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-H82E6-25 ug, ACRO Biosystems®) is added to each well and incubated again at 37°C for 1 hour. Next, the 96-well plate is washed three times using the washing buffer, and 100 μL of Streptavidin-HRP coupling in dilution buffer is added and incubated at 37°C for 1 hour. Next, the 96-well plate is washed, and each well is incubated with 200 μL of TMB (3,3',5,5'-tetramethyl-benzidine) for 20 minutes under light-shielded conditions at 37°C. Then, 50 μL of stop solution is added to halt 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 produced by transduction of a VSV-G pseudotyped lentivirus carrying the human ACE2 gene.

[0037] Plasmid for SARS-CoV-2 spike expression

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

[0039] Alpha mutants (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 mutant strains (B.1.617.2): T19R, G142D, 156-157 del, R158G, L452R, T478K, D614G, P681R, D950N.

[0043] Omicron mutant strains (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, L981F.

[0045] Omicron subtype mutants 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 possessing the SARS-CoV-2 spike protein are generated by 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 plasmids are delivered to the cells using the TransIT-LT1 transfection reagent. The medium is changed at 16 hours, and harvested at 48 and 72 hours after transfection. Cell debris is removed by centrifugation, and the supernatant is passed through a 0.45 μm injection filter. The pseudotyped lentiviruses are divided and stored at -80°C.

[0049] Evaluation of lentivirus titer by cell viability tests

[0050] In some embodiments, the introduced unit (TU) of SARS-CoV-2 pseudotyped lentivirus is evaluated by cell viability testing to reflect the dilution limit of the lentivirus. Briefly, HEK-293T cells stably expressing the human ACE2 gene are inoculated into a 96-well plate the day before lentivirus introduction. Different amounts of lentivirus are added to a medium containing polybren for titration of the pseudotyped lentivirus. The 96-well plate is rotated and infected. After culturing the cells at 37°C, the medium containing the virus and polybren is removed and replaced with fresh, complete DMEM containing primycin. After primycin treatment, the medium is removed and cell viability is detected with 10% AlarmaBlue reagent according to the manufacturer's instructions. The viability of uninfected cells (without primycin treatment) is 100%. The viral titer (introduced unit) is determined by plotting the doses of the diluted virus against the viable cells.

[0051] Neutralization test of pseudotyped lentivirus

[0052] In some embodiments, for the virus neutralization test, heat-inactivated serum is serially diluted to the desired dilution concentration, with the extract or single compound added simultaneously, and cultured with 1,000 TU of SARS-CoV-2 pseudotyped lentivirus in DMEM (with 1% FBS and 100 U / mL penicillin / streptomycin added) at 37°C for 1 hour. Then, 10,000 HEK-293T cells stably expressing the human ACE2 gene are inoculated into a 96-well plate. Sixteen hours after infection, the medium is replaced with fresh complete DMEM (with 10% FBS and 100 U / mL penicillin / streptomycin added), and the cells are cultured separately for 48 hours before the luciferase assay. For the luciferase assay, the expression level of the luciferase gene is measured by the Bright-Glo® luciferase assay system. Relative luminal units (RLU) are detected by Molecular Devices-SpectraMaxL. The method for calculating the suppression percentage is the ratio of the decrease 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 That is the case.

[0053] The following provides further details on the citrus extracts of this disclosure and their uses in the treatment of COVID-19, citrus extracts and test examples, but these are for illustrative purposes only and are not intended to limit this disclosure. The scope of protection of this disclosure shall be based on the content specified in the subsequent claims.

[0054] Examples

[0055] The methods disclosed herein will be described below in terms of a series of operations and steps, but the order in which these operations and steps are shown should not be construed as a limitation on this disclosure. For example, some operations or steps may be performed in a different order and / or simultaneously with other steps. Notwithstanding that all operations, steps and / or features must be performed to achieve the embodiments of this disclosure. Furthermore, each of the operations or steps described herein may include multiple sub-steps or operations.

[0056] To clarify, features and elements that are known in the field and are not necessary to understand the explained principles may be omitted.

[0057] Example 1: Preparation of the extract

[0058] Please refer to Figure 1, which is a flowchart illustrating a method for preparing a Shikuwasa extract according to one embodiment of the present disclosure.

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

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

[0061] Next, the ethyl acetate layer extract was taken and separated from the bioactive substances in the ethyl acetate layer extract using medium-pressure liquid chromatography (MPLC) on a silica gel column (SNAP Cartridge KP-Sil 340g, Biotage®). The medium-pressure liquid chromatography generated 110 layers (Fr.1 to Fr.110) using hexane (hexane) as the mobile phase, ethyl acetate (100:0 to 0:100), and acetone.

[0062] Next, using a silica gel column (Luna® silica column 5μm, 250×10mm, Phenomenex®), 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).

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

[0064] Layer Fr.59 generated 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 are obtained by first using layer Fr.66 as a silica gel column (SNAP Cartidge KP-Sil 10g, Biotage®) and a mobile phase of hexane and ethyl acetate (100:0-0:100), and SubFr.66-14 is a flavones compound (Cd7).

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

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

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

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

[0070] Example 2: Analysis of the extract

[0071] The crude extract, ethyl acetate layer extract, n-butanol layer extract, and aqueous layer 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 high-performance liquid chromatography (FLA) chromatogram of the crude extract, with absorbance values ​​of 220 nm, 280 nm, 320 nm, and 400 nm. Figure 3 is a FLA chromatogram of the ethyl acetate layer extract. Figure 4 is a FLA chromatogram of the n-butanol layer extract. Figure 5 is a FLA chromatogram of the aqueous layer extract. Figure 6 is an LC chromatogram of the crude extract obtained using a combination of liquid chromatography-mass spectrometry, with the upper row representing positive charges and the lower row representing negative charges. Figures 7 to 13 follow with the same arrangement. Figure 7 is an MS chromatogram of the crude extract obtained using a combination of liquid chromatography-mass spectrometry, with its mass-to-charge ratio (m / z) ranging from 100 to 1500. Figures 9, 11, and 13 follow with the same arrangement. Figure 8 shows the LC chromatogram of the ethyl acetate layer extract using liquid chromatography-mass spectrometry. Figure 9 shows the MS chromatogram of the ethyl acetate layer extract using liquid chromatography-mass spectrometry. Figure 10 shows the LC chromatogram of the n-butanol layer extract using liquid chromatography-mass spectrometry. Figure 11 shows the MS chromatogram of the n-butanol layer extract using liquid chromatography-mass spectrometry. Figure 12 shows the LC chromatogram of the aqueous layer extract using liquid chromatography-mass spectrometry. Figure 13 shows the MS chromatogram of the aqueous layer extract using liquid chromatography-mass spectrometry.

[0073] Example 3 Compound Identification

[0074] NMR assays were performed on the above 10 compounds.

[0075] Cd1, furo[3,2-g]chromen-7-one (psoralen). 1H: 6.36 (d, J = 9.6 Hz, 1H) 6.81 (dd, J = 2.3, 1.0 Hz, 1H) 7.46 (d, J = 1.1 Hz, 1H) 7.67 (s, 1H) 7.68 (d, J = 2.3 Hz, 1H) 7.78 (d, J = 9.6 Hz, 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, Bergapten). 1 H: 4.24 (s, 3H) 6.24 (d, J = 9.8 Hz, 1H) 6.99 (dd, J = 2.4, 1.0 Hz, 1H) 7.10 (t, J = 1.0 Hz, 1H) 7.57 (d, J = 2.4 Hz, 1H) 8.12 (dd, J = 9.8, 1.0 Hz, 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] Cd3,2’ - 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 = 8 Hz, 2H) 7.45 (d, J = 15.8 Hz, 1H) 7.59 (d, J = 15.8 Hz, 1H) 7.68 (d, J = 8 Hz, 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] Cd4,2'-Hydroxy-3',4,4',5',6'-pentamethoxy chalcone(E). 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] Cd5,2'-Hydroxy-3,4,4',5',6'-pentamethoxychalcone(E) 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] Cd6,2'-Hydroxy-3,3',4,4',5',6'-Hexamethoxychalcone(E) 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] Cd7,5-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-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. 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 structural formulas of 10 types of compounds JPEG0007872955000001.jpg139114

[0085] Example 4: Inhibitory ability of the extract to inhibit the binding of the novel coronavirus to ACE2.

[0086] The inhibitory performance of four types of extracts on the binding of SARS-CoV-2 spike protein wild-type (WT, Wuhan strain) or mutant strains (α, β, δ, or ο) to biotinylated human ACE2 recombinant protein was evaluated using enzyme-linked immunosorbent assay (ISIS). The experiments were mainly divided into a control group (Blank; B), a control group (Ctrl.; also called the ACE2 group, i.e., containing only ACE2 and virus, without any extracts), an inhibitor group (Inhibitor, Inh., 10 μg / mL, cat.GTX635791, GeneTex®, which recognizes full-length SARS-CoV-2 RBD recombinant protein (Wuhan-Hu-1 strain)), and a group of various extracts (e.g., crude extract, ethyl acetate layer extract, n-butanol layer extract, aqueous layer extract).

[0087] Please refer to Figure 14, which is a bar graph showing the binding rates of the novel coronavirus strain and ACE2 for the four types of extracts (5 mg / mL) from 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 strain to ACE2.

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

[0089] Please refer to Figure 16, which is a bar graph showing the binding rate of the SARS-CoV-2 A1pha mutant to 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 suppressed the binding of the SARS-CoV-2 A1pha mutant to ACE2.

[0090] Please refer to Figure 17, which is a bar graph showing the binding rate of the SARS-CoV-2 A1pha mutant 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 suppress the binding of the SARS-CoV-2 A1pha mutant to ACE2.

[0091] Please refer to Figure 18, which is a bar graph showing the binding rate of the SARS-CoV-2 A1pha mutant to ACE2 using the aqueous extract from Example 1. As can be seen from the results, the aqueous extracts at each concentration did not clearly inhibit the binding of the SARS-CoV-2 A1pha mutant to ACE2.

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

[0093] Please refer to Figure 20, which is a bar graph showing the binding rate of the Beta variant of the novel coronavirus to 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 suppressed the binding of the Beta variant of the novel coronavirus to ACE2.

[0094] Please refer to Figure 21, which is a bar graph showing the binding rate of the Beta variant of the novel coronavirus 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 significantly suppressed the binding of the Beta variant of the novel coronavirus to ACE2, and the n-butanol layer extracts at concentrations of 0.25 mg / mL to 0.5 mg / mL also tended to suppress it to some extent.

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

[0096] Please refer to Figure 23, which is a bar graph showing the binding rate of the SARS-CoV-2 Delta mutant to ACE2 using the crude extract from Example 1. As can be seen from the results, crude extracts with concentrations of 1.25 mg / mL to 10 mg / mL significantly suppressed the binding of the SARS-CoV-2 Delta mutant to ACE2.

[0097] Please refer to Figure 24, which is a bar graph showing the binding rate of the SARS-CoV-2 Delta mutant to 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 1.25 mg / mL to 10 mg / mL significantly suppressed the binding of the SARS-CoV-2 Delta mutant to ACE2.

[0098] Please refer to Figure 25, which is a bar graph showing the binding rate of the n-butanol layer extract of Example 1 to the SARS-CoV-2 Delta mutant and ACE2. 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 suppress the binding of the SARS-CoV-2 Delta mutant to ACE2.

[0099] Please refer to Figure 26, which is a bar graph showing the binding rate of the SARS-CoV-2 Delta mutant to ACE2 using the aqueous extract from Example 1. As can be seen from the results, the aqueous extract did not clearly inhibit the binding of the SARS-CoV-2 Delta mutant 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 SARS-CoV-2 Delta mutant using the crude extract from Example 1. As can be seen from the results, crude extracts with concentrations of 1.25 mg / mL to 10 mg / mL significantly suppressed the binding between the RBD region and ACE2 in the SARS-CoV-2 Delta mutant.

[0101] Please refer to Figure 28, which is a bar graph showing the binding rate between the RBD region and ACE2 in the SARS-CoV-2 Delta mutant 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 suppressed the binding between the RBD region and ACE2 in the SARS-CoV-2 Delta mutant.

[0102] Please refer to Figure 29, which is a bar graph showing the binding rate between the RBD region and ACE2 in the SARS-CoV-2 Delta mutant using the n-butanol layer extract of Example 1. As can be seen from the results, n-butanol layer extracts at concentrations of 1.25 mg / mL to 5 mg / mL tended to or significantly suppressed the binding between the RBD region and ACE2 in the SARS-CoV-2 Delta mutant.

[0103] Please refer to Figure 30, which is a bar graph showing the binding rate between the RBD region and ACE2 in the SARS-CoV-2 Delta mutant using the aqueous extract from Example 1. As can be seen from the results, the aqueous extract at each concentration did not clearly suppress the binding between the RBD region and ACE2 in the SARS-CoV-2 Delta mutant.

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

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

[0106] Please refer to Figure 33, which is a bar graph showing the binding rates of the Omicron BA.4 / 5 mutant of the novel coronavirus to ACE2 using four different extracts from several embodiments of this 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 layer extract, n-butanol layer extract, and aqueous layer extract all significantly inhibited the binding of the Omicron BA.4 / 5 mutant of the novel coronavirus to ACE2. Also, as can be seen from the figure, the inhibitors did not effectively inhibit the binding of the Omicron BA.4 / 5 mutant of the novel coronavirus to ACE2.

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

[0108] For the virus neutralization test, infection was induced by inoculating 10,000 HEK-293T cells stably expressing the human ACE2 gene with 1,000 TU SARS-CoV-2 pseudotyped lentivirus in a 96-well plate. Simultaneously, different extracts or compounds from Example 1 at different concentrations were added, and the binding of the virus to ACE2 was observed. The half-limiting IC50 concentration was then determined. 50 It was recorded.

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

[0110] Please refer to Figure 35, which shows the binding rate of the crude extract of Example 1 to the SARS-CoV-2 Omicron BA.4 / 5 mutant and ACE2. As can be seen from the results, the half-limiting IC50 is... 50 The concentration was 77.4 μg / mL.

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

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

[0113] Please refer to Figures 38A-38J, which show the binding rates of compounds Cd1-Cd10 from Example 1 to the Omicron BA.1 mutant strain of the novel coronavirus and ACE2. As can be seen from the results, the half-number inhibitory concentrations IC of Cd1-Cd10 50The concentrations were 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-39J, which show the binding rates of compounds Cd1-Cd10 from Example 1 to the Omicron BA.2 mutant strain of the novel coronavirus and ACE2. As can be seen from the results, the half-number inhibitory concentrations IC of Cd1-Cd10 50 The concentrations 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-40J, which show the binding rates of compounds Cd1-Cd10 from Example 1 to the Omicron BA.4 / 5 mutant strain of the novel coronavirus and ACE2. As can be seen from the results, the half-number inhibitory concentrations IC of Cd1-Cd10 50 The concentrations 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, ethyl acetate layer extract, n-butanol layer extract, and aqueous layer extract each have the effect of inhibiting the binding of the novel coronavirus-derived strain to ACE2.

[0117] In some embodiments, crude extracts and ethyl acetate layer extracts have the effect of inhibiting the binding of the A1pha variant of the novel coronavirus to ACE2.

[0118] In some embodiments, crude extracts, ethyl acetate layer extracts, and n-butanol layer extracts containing more than 2 mg / mL, and aqueous layer extracts containing more than 0.5 mg / mL, have the effect of suppressing the binding of the SARS-CoV-2 Beta mutant to ACE2.

[0119] In some embodiments, crude extracts, ethyl acetate layer extracts, and n-butanol layer extracts containing 1.25 to 2.5 mg / mL have the effect of suppressing the binding of the SARS-CoV-2 Delta mutant to ACE2.

[0120] In some embodiments, crude extracts, ethyl acetate layer extracts, and n-butanol layer extracts containing 1.25 to 5 mg / mL have the effect of suppressing the binding of the RBD region of the SARS-CoV-2 Delta mutant to ACE2.

[0121] In some embodiments, crude extracts, ethyl acetate layer extracts, and n-butanol layer extracts have the effect of inhibiting the binding of SARS-CoV-2 BA.1 and BA.2 mutants to ACE2.

[0122] In some embodiments, the crude extract, ethyl acetate layer extract, n-butanol layer extract, and aqueous layer extract have the effect of inhibiting the binding of the SARS-CoV-2 BA.4 / 5 mutant to ACE2.

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

[0124] While embodiments are disclosed as described above, this does not limit the disclosure, and anyone skilled in the art can make various changes and modifications as long as they do not deviate from the spirit and scope of the disclosure. Accordingly, the scope of protection of this disclosure shall be limited to what is specified in the subsequent claims.

Claims

1. The process of providing the shikwasa fruit, The process involves extracting the aforementioned shikwasa fruit with 95% ethanol to obtain a liquid portion and the extracted fruit, The process involves drying the aforementioned liquid portion to obtain a crude extract, The crude extract is partitioned with ethyl acetate and water to obtain an ethyl acetate layer and a highly polar layer, and the ethyl acetate layer is dried to obtain an ethyl acetate extract. The process involves partitioning the highly polar layer with n-butanol and water to obtain an n-butanol layer and an aqueous layer, drying the n-butanol layer to obtain an n-butanol extract, and drying the aqueous layer to obtain an aqueous layer extract. A composition for inhibiting the novel coronavirus, comprising the crude extract, the ethyl acetate extract, the n-butanol extract, the aqueous layer extract, or a combination thereof, prepared by any of the following steps.

2. The ethyl acetate extract comprises psoralen, 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, according to claim 1, for suppressing the novel coronavirus.

3. The composition for suppressing the novel coronavirus according to claim 1, wherein the step of extracting the shikwasa fruit with 95% ethanol comprises extracting the shikwasa fruit and ethanol in a weight-to-volume ratio of 1:5 to 15 (w / v).

4. The composition for suppressing the novel coronavirus according to claim 1, further comprising a pharmaceutically acceptable carrier.

5. The composition for suppressing the novel coronavirus according to claim 1, wherein the novel coronavirus comprises the origin strain, alpha mutant strain, beta mutant strain, gamma mutant strain, delta mutant strain, omicron mutant strain, or a combination thereof.

6. The composition for suppressing the novel coronavirus according to claim 5, wherein the omicron mutant strain includes a BA.1 subtype mutant, a BA.2 subtype mutant, a BA.3 subtype mutant, a BA.4 subtype mutant, a BA.5 subtype mutant, or a combination thereof.

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