Citrus extract and its use for inhibiting sars-cov-2

Abstract

The present application provides a citrus extract, after being extracted by ethanol, ethyl acetate, and n-butanol, a plurality of extracts are obtained, wherein a plurality of compounds are separated from the ethyl acetate extract. The plurality of extracts and the plurality of compounds all have the effect of inhibiting the novel coronavirus.

Description

Technical Field

[0001] This disclosure relates to an extract, a compound and its use, and in particular to a citrus extract, a compound and its use in 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 began to mutate from the original strain in 2019, with the Omicron variant becoming the dominant circulating strain in 2022. Although the Omicron variant causes a lower rate of severe illness, and rapid antigen testing and next-generation vaccines have been introduced, the COVID-19 pandemic has not yet subsided.

[0003] Therefore, there is a real need to improve existing technologies in order to provide natural and effective ways to combat the COVID-19 virus. Summary of the Invention

[0004] One embodiment of this disclosure provides a citrus extract prepared by the following steps: 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, wherein the ethyl acetate layer is dried to obtain an ethyl acetate extract; and partitioning the highly polar layer with n-butanol and water to obtain an n-butanol layer and an aqueous layer, wherein the n-butanol layer is dried to obtain an n-butanol extract, and the aqueous layer is dried to obtain an aqueous 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-tetramethoxyflavonoid, 5-hydroxy-3',4',6,7,8-pentamethoxyflavonoid, 3',4',5,6,7,8-hexamethoxyflavonoid, and 3',4',5,7,8-pentamethoxyflavanone.

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

[0007] Another embodiment of this disclosure provides a compound comprising 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 a pharmaceutically acceptable salt or ester thereof.

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

[0009] In some embodiments, the composition also includes a pharmaceutically acceptable carrier.

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

[0011] In some implementations, the omicron variant includes BA.1 subtype variants, BA.2 subtype variants, BA.3 subtype variants, BA.4 subtype variants, BA.5 subtype variants, or combinations thereof.

[0012] Another embodiment of this disclosure provides the use of the citrus extract as described above for the preparation of a drug to inhibit the novel coronavirus, wherein the citrus extract comprises a crude extract, an ethyl acetate extract, a n-butanol extract, or an aqueous extract.

[0013] Another embodiment of this disclosure provides the use of a compound for preparing a medicament to inhibit the novel coronavirus, wherein the compound comprises 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 combinations thereof.

[0014] In some implementations, inhibiting the novel coronavirus includes the original strain, alpha variant, beta variant, gamma variant, delta variant, omicron variant, or a combination thereof.

[0015] In some implementations, the omicron variant includes BA.1 subtype variants, BA.2 subtype variants, BA.3 subtype variants, BA.4 subtype variants, BA.5 subtype variants, or combinations thereof.

[0016] In some implementations, the drug is in the form of capsules, tablets, powders, or liquids.

[0017] In some implementations, the drug works by inhibiting the binding of the novel coronavirus to the ACE2 protein.

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

[0019] In some implementations, the disease is severe special infectious pneumonia (or COVID-19). Attached Figure Description

[0020] The various aspects of this disclosure will be most readily understood when read in conjunction with the accompanying drawings. It should be noted that, according to industry standard operating procedures, the various features may not be drawn to scale. In fact, for clarity of explanation, the dimensions of the various features can be arbitrarily increased or decreased. To make the above and other objects, features, advantages, and embodiments of this disclosure more apparent and understandable, the accompanying drawings are described below: Figure 1 A flowchart illustrating a method for preparing lemon extract from Taiwan, China, according to some embodiments disclosed herein.

[0021] Figure 2 The diagram illustrates high-performance liquid chromatography (HPLC) spectra of crude extracts from some embodiments disclosed herein.

[0022] Figure 3 The diagram illustrates the high-performance liquid chromatography (HPLC) spectra of ethyl acetate layer extracts from some embodiments disclosed herein.

[0023] Figure 4 The diagram illustrates the high-performance liquid chromatography (HPLC) spectra of n-butanol layer extracts according to some embodiments disclosed herein.

[0024] Figure 5 The diagram illustrates high-performance liquid chromatography (HPLC) spectra of aqueous extracts from some embodiments disclosed herein.

[0025] Figure 6 The illustration shows the LC spectra of crude extracts obtained by liquid chromatography-mass spectrometry (LC-MS) in some embodiments of the present invention.

[0026] Figure 7 MS spectra of crude extracts obtained by liquid chromatography-mass spectrometry in some embodiments disclosed herein are illustrated.

[0027] Figure 8 The LC spectra of ethyl acetate layer extracts in liquid chromatography-mass spectrometry (LC-MS) of some embodiments disclosed herein are illustrated.

[0028] Figure 9 MS spectra of ethyl acetate layer extracts from some embodiments disclosed herein are illustrated using liquid chromatography-mass spectrometry.

[0029] Figure 10 The LC spectra of n-butanol layer extracts in some embodiments disclosed herein are illustrated.

[0030] Figure 11 MS spectra of n-butanol layer extracts in liquid chromatography-mass spectrometry (LC-MS) of some embodiments disclosed herein are illustrated.

[0031] Figure 12 The LC spectra of aqueous extracts in some embodiments of this invention are illustrated.

[0032] Figure 13 MS spectra of aqueous extracts obtained in some embodiments of this invention are illustrated using liquid chromatography-mass spectrometry.

[0033] Figure 14 A bar chart illustrating the binding rates of four extracts from some of the embodiments disclosed herein to the original strain of SARS-CoV-2 and ACE2.

[0034] Figure 15 A bar chart illustrating the binding rates of crude extracts from some of the embodiments disclosed herein to SARS-CoV-2 A1pha variants and ACE2.

[0035] Figure 16 A bar chart illustrating the binding rates of ethyl acetate layer extracts of some embodiments disclosed herein to ACE2 and the A1pha variant of SARS-CoV-2.

[0036] Figure 17 A bar chart illustrating the binding rates of n-butanol layer extracts of some embodiments disclosed herein to ACE2 and the A1pha variant of SARS-CoV-2.

[0037] Figure 18 A bar chart illustrating the binding rates of aqueous extracts of some embodiments of the SARS-CoV-2 A1pha variant to ACE2 is presented.

[0038] Figure 19A bar chart illustrating the binding rates of crude extracts from some of the embodiments disclosed herein to ACE2 on the beta variant of SARS-CoV-2.

[0039] Figure 20 A bar chart illustrating the binding rate of ethyl acetate layer extracts of some embodiments disclosed herein to ACE2 of SARS-CoV-2 Beta variants.

[0040] Figure 21 A bar chart illustrating the binding rate of n-butanol layer extracts of some embodiments disclosed herein to ACE2 of the SARS-CoV-2 Beta variant.

[0041] Figure 22 A bar chart illustrating the binding rate of aqueous extracts of some embodiments of the disclosed invention to ACE2 for the Beta variant of SARS-CoV-2.

[0042] Figure 23 A bar chart illustrating the binding rates of crude extracts from some of the embodiments disclosed herein to ACE2 of the Delta variant of SARS-CoV-2.

[0043] Figure 24 A bar chart illustrating the binding rates of ethyl acetate layer extracts of some embodiments disclosed herein to ACE2 and the Delta variant of SARS-CoV-2.

[0044] Figure 25 A bar chart illustrating the binding rates of n-butanol layer extracts of some embodiments disclosed herein to ACE2 and the Delta variant of SARS-CoV-2.

[0045] Figure 26 A bar chart illustrating the binding rates of aqueous extracts of some embodiments of the SARS-CoV-2 Delta variant to ACE2 is presented.

[0046] Figure 27 A bar chart illustrating the binding rate of crude extracts from some of the embodiments disclosed herein to ACE2 in the RBD region (receptor-binding domain) of the SARS-CoV-2 Delta variant.

[0047] Figure 28 A bar chart illustrating the binding rate of ethyl acetate layer extracts of some embodiments disclosed herein to the RBD region of ACE2 in the Delta variant of SARS-CoV-2.

[0048] Figure 29 A bar graph illustrating the binding rate of n-butanol layer extracts of some embodiments disclosed herein to the RBD region of ACE2 in the Delta variant of SARS-CoV-2.

[0049] Figure 30A bar chart illustrating the binding rate of aqueous extracts of some embodiments of the present invention to the RBD region of ACE2 in the Delta variant of SARS-CoV-2 is shown.

[0050] Figure 31 A bar chart illustrating the binding rates of five extracts of the SARS-CoV-2 Omicron BA.1 variant to ACE2 in some of the embodiments disclosed herein.

[0051] Figure 32 A bar chart illustrating the binding rates of four extracts from some of the embodiments disclosed herein to the Omicron BA.2 variant of SARS-CoV-2 and ACE2.

[0052] Figure 33 A bar chart illustrating the binding rates of four extracts from some of the embodiments disclosed herein to ACE2 of the Omicron BA.4 / 5 variant of SARS-CoV-2.

[0053] Figure 34 The illustration shows the binding rate of crude extracts of some of the embodiments disclosed herein to the SARS-CoV-2 Omicron BA.2 variant and ACE2.

[0054] Figure 35 The illustration shows the binding rate of crude extracts of some of the embodiments disclosed herein to ACE2 of the Omicron BA.4 / 5 variant of SARS-CoV-2.

[0055] Figure 36 The illustration shows the binding rate of ethyl acetate layer extracts of some embodiments of the SARS-CoV-2 Omicron BA.2 variant to ACE2.

[0056] Figure 37 The illustration shows the binding rate of ethyl acetate layer extracts of some embodiments of the SARS-CoV-2 Omicron BA.4 / 5 variant to ACE2.

[0057] Figures 38A to 38J The illustration shows the binding rates of compounds Cd1 to Cd10 of some embodiments disclosed herein to ACE2 of the Omicron BA.1 variant of SARS-CoV-2.

[0058] Figures 39A to 39J The illustration shows the binding rates of compounds Cd1 to Cd10 of some embodiments disclosed herein to ACE2 on the Omicron BA.2 variant of SARS-CoV-2.

[0059] Figures 40A to 40J The illustration shows the binding rates of compounds Cd1 to Cd10 of some embodiments disclosed herein to ACE2 on the Omicron BA.4 variant of SARS-CoV-2. Detailed Implementation

[0060] To make the description of this disclosure more detailed and complete, illustrative descriptions of embodiments and specific examples of this disclosure are provided below. However, these are not the only forms of implementing or utilizing the specific examples of this disclosure. The various embodiments disclosed below can be combined or substituted with each other where advantageously possible, and other embodiments can be added to one embodiment without further description or explanation. In the following description, many specific details will be set forth in detail to enable the reader to fully understand the following embodiments. However, the embodiments of this disclosure can also be practiced without such specific details.

[0061] In this document, unless otherwise specified in the text, “a” and “the” may refer to one or more. It will be further understood that the terms “comprising,” “including,” “having,” and similar terms as used herein specify the features, regions, integers, steps, operations, elements, and / or components described herein, but do not exclude one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof described or additionally described herein.

[0062] In this article, the term "citrus" refers to the genus Citrus, which belongs to the family Rutaceae and includes both trees and shrubs. Some important fruits belong to this genus, such as citron (Citrus medica), pomelo (Citrus maxima), mandarin orange (Citrus reticulata), orange (Citrus sinensis), lemon (Citrus limon), lime (Citrus limonia), lime (Citrus aurantifolia), bitter orange (Citrus aurantium), sweet orange (Citrus junos), kumquat (Citrus japonica, Cj), grapefruit (Citrus paradisi), red river orange (Citrushongheensis), Yichang orange (Citrus ichangensis), wasp orange (Citrus macroptera var. kerrii), Thai lime (Citrus hystrix), tangerine (Citrus tachibana), and Taiwan lemon (Citrus depressa, Cd).

[0063] Taiwanese lemon, also known as flat lemon, is one of the native citrus varieties of Taiwan.

[0064] In this article, the term "fruit" refers to the fact that the structure of a fruit can generally be divided into two parts: the seed and the pericarp. The pericarp can be further divided into the exocarp, mesocarp, and endocarp. The exocarp is leathery, soft, and thick; the endocarp is a thick, juicy, fleshy part, also known as the pulp; the mesocarp is the tissue between the exocarp and the endocarp, and compared to the endocarp, the mesocarp mostly presents a white, compact structure.

[0065] In this article, furocoumarines refer to a series of compounds in which the 7-hydroxyl group of the parent nucleus condenses with the 6- or 8-substituted isopentenyl group to form a furan ring (position numbering is shown in formula (I) for example). Psoralen belongs to the linear furocoumarines, while bergapten is classified as an angular furocoumarine.

[0066] In some embodiments, the organic solvent includes, but is not limited to, polar and nonpolar organic solvents. For example, polar organic solvents include, but are not limited to, alcohols, ketones (acetone, dialkyl ketones, pyrollidones), 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, ethoxylated alcohols, polyethylene glycol, carboxylic acid esters, polyalkylglycol esters, anhydrous sorbitol, glycol esters, carboxylic acid amides, monoalkanolamines, polyethylene fatty acid amides, polysorbate esters, cyclodextrins, sugar-based alcohols, silicone-based alcohols, polyalkylated alcohols, and alkylaryl ethoxides.

[0067] In this article, the term "partition" refers to a liquid that separates into layers after mixing the solvent and the extractant. For example, mixing ethyl acetate and water (1:1 volume) with the extractant results in a liquid with a low-polarity layer and a high-polarity layer.

[0068] In this article, the term "COVID-19" refers to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as the novel coronavirus or 2019 novel coronavirus.

[0069] In this article, the term "medically acceptable salt" refers to a salt that retains the bioavailability and properties of a free base or free acid and is generally biologically or otherwise desirable.

[0070] In this article, the term "pharmaceutically acceptable ester" means that compounds of formulas (I) to (X) can be derivatized on functional groups to provide derivatives that can be converted back to the parent compound in vivo.

[0071] In some embodiments, pharmaceutically acceptable carriers include, but are not limited to, 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. Enhancers, anti-irritants, colorants, propellants, surfactants, and other similar or applicable carriers of the present invention.

[0072] Spike protein binding assay In some implementations, an enzyme-linked immunosorbent assay (ELISA) was used to evaluate the inhibitory efficacy of four extracts against the binding of wild-type (WT, Wuhan strain) or variant (α, β, γ, δ, or O) SARS-CoV-2 spike protein to biotinylated recombinant human ACE2 protein. First, 100 μL of spike protein was coated onto each well of a 96-well plate and diluted with buffer, which was incubated overnight at 4°C. The coated plate was then washed three times with a wash buffer consisting of PBS containing Tween-20, followed by blocking with 250 μL of blocking buffer containing fetal 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. ® Add the protein to a 96-well plate and incubate at 37°C for 1 hour. Then, add 100 μL of biotinylated human ACE2 protein (10 ng / mL; cat. AC2-H82E6-25ug; ACRO Biosystems). ® Add 100 μL of Streptavidin-HRP conjugate to each well and incubate at 37°C for 1 hour. Next, wash the 96-well plate three times with washing buffer, then add 100 μL of Streptavidin-HRP conjugate in dilution buffer and incubate at 37°C for 1 hour. Then, wash the 96-well plate and incubate at 37°C in the dark with 200 μL of TMB (3,3',5,5'-tetramethyl-benzidine) per well for 20 minutes. Finally, add 50 μL of stop solution to terminate the reaction, and measure the absorbance at 450 nm using a microdisc detector.

[0073] Cell culture In some embodiments, human embryonic kidney (HEK-293T / 17; ATCC) ® CRL-11268TM cells were obtained from the American Type Culture Collection (ATCC). Cells were cultured at 37°C in DMEM (Dulbecco's modified eagle medium) containing 10% fetal bovine serum and 1x penicillin-streptomycin. HEK-293T-ACE2 cells were produced by transduction of a VSV-G pseudotyped lentivirus carrying the human ACE2 gene.

[0074] Plasmids for SARS-CoV-2 spike expression In some implementations, the synthetic DNA fragment encoding the SARS-CoV-2 spike gene was purchased from Integrated DNA Technologies (IDT) and cloned into a mammalian expression vector. The mutation sites for each spike mutant are listed below: Alpha variant (B.1.1.7): 69-70 del, Y144 del, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H.

[0075] Beta variant (B.1.351 (501Y.V2)): L18F, D80A, D215G, 242-244 del, R246I, K417N, E484K, N501Y, D614G, A701V.

[0076] Gamma variants (Lineage P1): L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I.

[0077] Delta variant (B.1.617.2): T19R, G142D, 156-157 del, R158G, L452R, T478K, D614G, P681R, D950N.

[0078] Omicron mutant (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.

[0079] Omicron subtype mutant strain 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.

[0080] Omicron subtype mutant strain 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.

[0081] Omicron subtype mutant strains 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.

[0082] Production and purification of SARS-CoV-2 spike pseudotype lentivirus In some implementations, pseudolentiviruses 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 were seeded one day prior to transfection, and the indicator plasmid was delivered into the cells using the TransIT-LT1 transfection reagent. The culture medium was refreshed at 16 hours and harvested at 48 and 72 hours post-transfection. Cell debris was removed by centrifugation, and the supernatant was passed through a 0.45 μm injection filter. The pseudolentiviruses were aliquoted and then stored at -80°C.

[0083] Cell survival assays to assess lentiviral titers In some implementations, the introduction unit (TU) of SARS-CoV-2 pseudotyped lentivirus is assessed using a cell survival assay to reflect the dilution limit of the lentivirus. Briefly, one day prior to lentivirus introduction, HEK-293T cells stably expressing the human ACE2 gene are seeded in 96-well plates. For titrating pseudotyped lentivirus, varying amounts of lentivirus are added to a medium containing polybrene. Infection is performed by rotation in the 96-well plates. After culturing the cells at 37°C, the medium containing the virus and polybrene is removed and replaced with fresh, complete DMEM containing puromycin. Following puromycin treatment, the medium is removed, and cell viability is assessed using 10% AlarmaBlue reagent according to the manufacturer's instructions. Uninfected cells (not treated with puromycin) have a 100% viability. Viral titer (TU) is determined by plotting surviving cells against diluted virus doses.

[0084] pseudolentivirus neutralization test In some embodiments, for the virus neutralization assay, heat-inactivated serum was serially diluted to the desired concentration while simultaneously adding an extract or a single compound, and incubated with 1,000 TU of SARS-CoV-2 pseudotyped lentivirus in DMEM (with 1% FBS and 100 U / mL penicillin / streptomycin) for 1 hour at 37°C. Then, 10,000 stable HEK-293T cells expressing the human ACE2 gene were seeded in 96-well plates. Sixteen hours post-infection, the medium was replaced with fresh, complete DMEM (with 10% FBS and 100 U / mL penicillin / streptomycin), and the cells were continuously cultured for an additional 48 hours before the luciferase assay. For the luciferase assay, the expression level of the luciferase gene was determined using a Bright-Glo™ luciferase assay system. Relative optical units (RLU) were detected by Molecular Devices-SpectraMaxL. The percentage of inhibition is calculated as the ratio of the decrease in RLU in the presence of diluted serum to the RLU in the serum-free control. The formula is as follows: (RLU) 对照 -RLU 血清 ) / RLU 控制 .

[0085] The following examples and experimental cases illustrate the citrus extract disclosed herein and its use in treating COVID-19 in more detail. However, they are for illustrative purposes only and are not intended to limit the scope of the disclosure. The scope of protection of the disclosure shall be defined by the appended claims.

[0086] Example Although the methods disclosed herein are illustrated using a series of operations or steps, the order in which these operations or steps are shown should not be construed as a limitation of this disclosure. For example, some operations or steps may be performed in a different order and / or simultaneously with other steps. Furthermore, it is not necessary to perform all illustrated operations, steps, and / or features to achieve the implementation of this disclosure. In addition, each operation or step described herein may comprise several sub-steps or actions.

[0087] For clarity, features and elements that are known in the domain and are not essential for understanding the principles described will be omitted.

[0088] Example 1 Preparation of extract Please see Figure 1 As shown, Figure 1 A flowchart illustrating a method for preparing Taiwanese lemon extract according to one embodiment of the present invention is shown.

[0089] Whole Taiwanese lemons were extracted with 95% ethanol at a weight-to-volume ratio of 1:10 (w / v) for one week, yielding a first liquid and a first extracted lemon. The first liquid portion was removed and evaporated under vacuum to obtain a dried first crude extract. The first extracted lemon was then extracted with 95% ethanol at a weight-to-volume ratio of 1:10 (w / v) for another week, yielding a second liquid and a second extracted lemon. The second liquid portion 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.

[0090] Next, the crude extract was partitioned three times with ethyl acetate and distilled water (1:1 ratio) to obtain an ethyl acetate layer (low polarity layer) and a high polarity layer. The high polarity layer was then partitioned three times with n-butanol and distilled water (1:1 ratio) to obtain 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.

[0091] Next, the ethyl acetate extract was subjected to medium-pressure liquid chromatography (MPLC) using a silica gel column (SNAP Cartridge KP-Sil 340g, Biotage). ® Bioactive substances were separated from the ethyl acetate layer extract using medium-pressure liquid chromatography with a mobile phase of hexane and ethyl acetate (100:0 to 0:100) and acetone, producing 110 layers (Fr.1 to Fr.110).

[0092] Next, use silicone pillars (Luna)® silica column 5 μm, 250×10 mm, Phenomenex ® The active components were selected by semi-preparative refractive index-high performance liquid chromatography (semi-preparative RI-HPLC) at the following intervals: Fr.44, Fr.59, Fr.66, and Fr.78.

[0093] Layered Fr.44 uses a mobile phase consisting of hexane and ethyl acetate (8:2) to produce two furanocoumarin compounds (Cd1, Cd2).

[0094] Layered Fr.59 uses a mobile phase consisting of hexane, ethyl acetate, and dichloromethane (7:1.5:1.5) to produce two chalcones (Cd3 and Cd4).

[0095] Sub-layered Fr.66-1 to Sub-Fr.66-18 involves first passing the layered Fr.66 through a silicone column (SNAPCartidge KP-Sil 10g, Biotage). ® It was obtained with a mobile phase of hexane and ethyl acetate (100:0-0:100), wherein SubFr.66-14 is a flavonoid compound (Cd7).

[0096] Sub-stratification SubFr.66-9 produced two chalcone compounds (Cd5, Cd6) from a mobile phase of hexane and ethyl acetate (7.7:2.3).

[0097] Sub-stratification SubFr.66-12 produces a flavonoid (Cd8) from a mobile phase of hexane and ethyl acetate (6.5:3.5).

[0098] Sub-stratification SubFr.66-13 produces a flavonoid (Cd10) from a mobile phase of hexane and ethyl acetate (6.3:3.7).

[0099] The layered Fr.78 used a mobile phase consisting of hexane and ethyl acetate (2.5:7.5) to produce a flavonoid compound (Cd9). The separation process described above was repeated to obtain the amount required for the activity experiments.

[0100] Example 2 Analysis of the extract 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.

[0101] Experimental results are as follows Figures 2 to 13 As shown. Figure 2 The spectrum is obtained by high-performance liquid chromatography of the crude extract, with absorbance values ​​at 220 nm, 280 nm, 320 nm, and 400 nm. Figure 3 This is a high-performance liquid chromatography (HPLC) chromatogram of the ethyl acetate layer extract. Figure 4 This is a high-performance liquid chromatography (HPLC) chromatogram of the n-butanol layer extract. Figure 5 This is a spectrum obtained by high-performance liquid chromatography (HPLC) of aqueous extracts. Figure 6 This is the LC spectrum of the crude extract obtained by liquid chromatography-mass spectrometry (LC-MS). The top row represents positively charged molecules, and the bottom row represents negatively charged molecules. (The following...) Figures 7 to 13 They are all arranged in the same way. Figure 7 The MS spectra of the crude extract obtained by liquid chromatography-mass spectrometry (LC-MS) show mass-to-charge ratios (m / z) ranging from 100 to 1500. Figure 9 , Figure 11 and Figure 13 They are all arranged in the same way. Figure 8 This is the LC spectrum of the ethyl acetate layer extract obtained by liquid chromatography-mass spectrometry. Figure 9 MS spectrum of ethyl acetate layer extract obtained by liquid chromatography-mass spectrometry. Figure 10 This is the LC spectrum of the n-butanol layer extract obtained by liquid chromatography-mass spectrometry. Figure 11 This is the MS spectrum of the n-butanol layer extract obtained by liquid chromatography-mass spectrometry. Figure 12 The LC spectrum is obtained from the aqueous extract using liquid chromatography-mass spectrometry. Figure 13 MS spectrum of aqueous extract obtained by liquid chromatography-mass spectrometry.

[0102] Example 3 Identification of Compounds NMR analysis was performed on the above ten compounds.

[0103] Cd1,furo[3,2-g]chromen-7-one (psoralen). 1 H: 6.36 (d, J = 9.6Hz, 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.

[0104] Cd2,4-methoxyfuro[3,2-g]chromen-7-one (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.

[0105] Cd3,2'-hydroxy-4,4',5',6'-tetramethoxychalcone (E) 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). 13C: 55.47 56.13 60.76 61.64 96.52 110.37 114.05114.70 115.31 118.90 124.67 124.75 128.30 129.44 130.47 134.76 143.56 153.30158.03 158.15 161.44 192.61.

[0106] Cd4,2'-hydroxy-3',4,4',5',6'-pentamethoxychalcone (E)2'-Hydroxy-3',4,4',5',6'-pentamethoxychalcone. 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 = 8.8 Hz, 2H) 7.14 (d, J = 16.0 Hz, 1H) 7.42 (d, J = 16.0Hz, 1H) 7.69 (d, J = 8.8 Hz, 2H). 13 C: 55.99 60.81 60.92 61.03 61.53 114.51116.23 125.50 126.91 130.47 137.13 138.50 144.21 146.31 147.28 149.23 161.37192.73.

[0107] Cd5,2'-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 = 8.3 Hz, 1H) 7.13 (d, J = 2.0 Hz, 1H) 7.21(dd, J = 8.3, 2.0 Hz, 1H) 7.77 (d, J = 15.6 Hz, 1H) 7.81 (d, J = 15.6 Hz, 1H).13 C:56.11 56.17 56.25 61.47 62.10 96.82 108.92 110.56 111.40 123.12 124.46 128.58135.46 143.79 149.43 151.50 155.08 160.16 162.82 192.89.

[0108] Cd6,2'-hydroxy-3,3',4,4',5',6'-hexamethoxy-chalcone (E) 1 H: 3.84 (s, 3H) 3.87 (s, 6H) 3.91 (s, 3H) 3.92 (s, 3H) 4.07 (s, 3H) 6.88 (d, J = 8.3 Hz, 1H) 7.13 (d, J = 2.0 Hz, 1H) 7.23 (dd, J =8.3, 2.0 Hz, 1H) 7.77 (d, J = 15.5 Hz, 1H) 7.81 (d, J = 15.5 Hz, 1H). 13C: 56.1456.20 61.23 61.52 61.80 62.39 110.60 111.31 111.41 123.33 128.41 137.49138.62 144.38 149.47 151.02 151.68 153.51 155.12 193.69.

[0109] 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 = 8.5 Hz, 1H) 7.41 (d, J = 2.1 Hz, 1H) 7.57(dd, J = 8.5, 2.1 Hz, 1H). 13C: 56.22 56.34 56.56 61.79 95.98 104.28 105.03109.06 111.50 120.38 124.04 129.17 149.58 149.64 152.63 157.79 158.82 164.06182.85.

[0110] 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 = 8.4 Hz, 1H) 7.40 (d, J = 2.1 Hz, 1H) 7.56 (dt, J = 8.4, 2.1 Hz, 1H). 13 C: 56.20 56.32 61.32 61.91 62.25 104.16 107.17 109.00 111.49120.37 123.88 133.15 136.78 145.97 149.59 149.72 152.69 153.20 164.15 183.18.

[0111] Cd9,3',4',5,6,7,8-hexamethoxyflavone (Nobiletin). ¹H: 3.86 (s, 9H) 3.88 (s, 3H) 3.94 (s, 3H) 4.02 (s, 3H) 6.52 (s, 1H) 6.90 (d, J = 8.5 Hz, 1H) 7.32 (d, J = 2.1 Hz, 1H) 7.47 (dd, J = 8.5, 2.1Hz, 1H). 13 C: 55.99 56.09 61.66 61.81 61.96 62.25 106.82 108.63 111.31 114.83119.67 123.99 138.04 144.10 147.73 148.39 149.32 151.45 151.99 161.10 177.36206.96.

[0112] Cd10,3',4',5,7,8-pentamethoxyflavanone. 1 H: 2.70 (dd, J = 16.6, 2.8 Hz, 1H) 2.97 (dd, J = 16.6, 13.4 Hz, 1H) 3.75 (s,3H) 3.81 (s, 3H) 3.83 (s, 3H) 3.85 (s, 3H) 3.87 (s, 3H) 5.28 (dd, J = 13.4, 2.8Hz, 1H) 6.29 (s, 1H) 6.84 (d, J = , 8.0 Hz, 1H) 6.92 (dd, J = 2.0, 8.0 Hz, 1H)6.93 (d, J = 2.0 Hz, 1H).

[0113] Table 1. Chemical structural formulas of ten compounds

[0114] Example 4: Inhibitory effect of extract on the binding of SARS-CoV-2 to ACE2 An enzyme-linked immunosorbent assay (ELISA) was used to evaluate the inhibitory efficacy of four extracts on the binding of wild-type (WT, Wuhan strain) or variant (α, β, δ, or ο) SARS-CoV-2 spike protein to biotinylated recombinant human ACE2 protein. The experiment consisted of a control group (Blank, B), a control group (Ctrl., or ACE2 group, i.e., containing only ACE2 and virus but without extracts), and an inhibitor group (Inhibitor, Inh., 10 μg / mL; cat. GTX635791, GeneTex). ® The study identified the full-length SARS-CoV-2 RBD recombinant protein (Wuhan-Hu-1 strain) and various extract groups (such as crude extract, ethyl acetate layer extract, n-butanol layer extract, and aqueous layer extract).

[0115] Please see Figure 14 , Figure 14 The bar chart shows the binding rates of the four extracts (5 mg / mL) from Example 1 to the original SARS-CoV-2 virus and ACE2. The results showed that the crude extract, ethyl acetate extract, n-butanol extract, and aqueous extract all significantly inhibited the binding of the original SARS-CoV-2 virus to ACE2.

[0116] Please see Figure 15 , Figure 15 This is a bar chart showing the binding rate of the crude extract of Example 1 to SARS-CoV-2 A1pha variant and ACE2. The results show that the crude extract at concentrations of 0.5 mg / mL to 2 mg / mL significantly inhibited the binding of SARS-CoV-2 A1pha variant to ACE2.

[0117] Please see Figure 16 , Figure 16 This is a bar chart showing the binding rate of the ethyl acetate extract of Example 1 to ACE2 in the A1pha variant of SARS-CoV-2. The results show that ethyl acetate extracts at concentrations from 0.25 mg / mL to 2 mg / mL significantly inhibited the binding of the A1pha variant of SARS-CoV-2 to ACE2.

[0118] Please see Figure 17 , Figure 17 This is a bar chart showing the binding rate of the n-butanol layer extract of Example 1 to ACE2 of the SARS-CoV-2 A1pha variant. The results show that the n-butanol layer extract at a concentration of 2 mg / mL tends to inhibit the binding of the SARS-CoV-2 A1pha variant to ACE2.

[0119] Please see Figure 18 , Figure 18 This is a bar chart showing the binding rate of the aqueous extract of Example 1 to the SARS-CoV-2 A1pha variant and ACE2. The results show that the aqueous extract at all concentrations did not significantly inhibit the binding of the SARS-CoV-2 A1pha variant to ACE2.

[0120] Please see Figure 19 , Figure 19 The bar chart shows the binding rate of the crude extract of Example 1 to ACE2 by the Beta variant of SARS-CoV-2. The results show that the crude extracts at concentrations of 0.5 mg / mL and 2 mg / mL significantly inhibited the binding of the Beta variant of SARS-CoV-2 to ACE2.

[0121] Please see Figure 20 , Figure 20 This is a bar chart showing the binding rate of the ethyl acetate extract of Example 1 to ACE2 in the Beta variant of SARS-CoV-2. The results show that ethyl acetate extracts at concentrations ranging from 0.25 mg / mL to 2 mg / mL significantly inhibited the binding of the Beta variant of SARS-CoV-2 to ACE2.

[0122] Please see Figure 21 , Figure 21A bar chart illustrating the binding rate of the n-butanol layer extract of Example 1 to ACE2 of the SARS-CoV-2 Beta variant is shown. The results show that the n-butanol layer extract at a concentration of 2 mg / mL significantly inhibited the binding of the SARS-CoV-2 Beta variant to ACE2; there was also a slight inhibitory trend from concentrations of 0.25 mg / mL to 0.5 mg / mL.

[0123] Please see Figure 22 , Figure 22 A bar chart illustrating the binding rate of the aqueous extract of Example 1 to ACE2 of the SARS-CoV-2 Beta variant is shown. The results indicate that aqueous extracts at concentrations from 0.5 mg / mL to 2 mg / mL significantly inhibited the binding of the SARS-CoV-2 Beta variant to ACE2.

[0124] Please see Figure 23 , Figure 23 A bar graph illustrating the binding rate of the crude extract of Example 1 to ACE2 of the SARS-CoV-2 Delta variant is shown. The results showed that the crude extract at concentrations from 1.25 mg / mL to 10 mg / mL significantly inhibited the binding of the SARS-CoV-2 Delta variant to ACE2.

[0125] Please see Figure 24 , Figure 24 A bar chart illustrating the binding rate of the ethyl acetate extract of Example 1 to ACE2 of the SARS-CoV-2 Delta variant is shown. The results indicate that ethyl acetate extracts at concentrations ranging from 1.25 mg / mL to 10 mg / mL significantly inhibited the binding of the SARS-CoV-2 Delta variant to ACE2.

[0126] Please see Figure 25 , Figure 25 A bar graph illustrating the binding rate of the n-butanol layer extract of Example 1 to ACE2 of the SARS-CoV-2 Delta variant is shown. The results indicate that n-butanol layer extracts at concentrations ranging from 1.25 mg / mL to 2.5 mg / mL tend to inhibit the binding of the SARS-CoV-2 Delta variant to ACE2.

[0127] Please see Figure 26 , Figure 26 A bar chart illustrating the binding rate of the aqueous extract of Example 1 to the SARS-CoV-2 Delta variant and ACE2 is shown. The results indicate that the aqueous extract did not significantly inhibit the binding of the SARS-CoV-2 Delta variant to ACE2.

[0128] Please see Figure 27 , Figure 27A bar graph illustrating the binding rate of the crude extract of Example 1 to the RBD region and ACE2 in the Delta variant of SARS-CoV-2 is shown. The results showed that the crude extract at concentrations from 1.25 mg / mL to 10 mg / mL significantly inhibited the binding of the RBD region and ACE2 in the Delta variant of SARS-CoV-2.

[0129] Please see Figure 28 , Figure 28 A bar chart illustrating the binding rate of the ethyl acetate extract of Example 1 to the RBD region and ACE2 in the Delta variant of SARS-CoV-2 is shown. The results indicate that ethyl acetate extracts at concentrations ranging from 1.25 mg / mL to 10 mg / mL significantly inhibited the binding of the RBD region and ACE2 in the Delta variant of SARS-CoV-2.

[0130] Please see Figure 29 , Figure 29 A bar graph illustrating the binding rate of the n-butanol layer extract of Example 1 to the RBD region and ACE2 in the Delta variant of SARS-CoV-2 is shown. The results indicate that n-butanol layer extracts at concentrations from 1.25 mg / mL to 5 mg / mL trended or significantly inhibited the binding of the RBD region and ACE2 in the Delta variant of SARS-CoV-2.

[0131] Please see Figure 30 , Figure 30 A bar chart illustrating the binding rate of the aqueous extract of Example 1 to the RBD region and ACE2 in the Delta variant of SARS-CoV-2 is shown. The results indicate that the aqueous extract at all concentrations did not significantly inhibit the binding of the RBD region and ACE2 in the Delta variant of SARS-CoV-2.

[0132] Please see Figure 31 , Figure 31 A bar chart is shown illustrating the binding rates of the four extracts from Example 1 to ACE2 of the SARS-CoV-2 Omicron BA.1 variant. The results showed that the crude extract (2 mg / mL), ethyl acetate extract, n-butanol extract, and aqueous extract all significantly inhibited the binding of the SARS-CoV-2 Omicron BA.1 variant to ACE2. Furthermore, the crude extract of kumquat (Cj) at 2 mg / mL, prepared using a method similar to that in Example 1 (extraction with ethanol), also significantly inhibited the binding of the SARS-CoV-2 Omicron BA.1 variant to ACE2. In addition, the layered Fr.82 promoted the binding of the SARS-CoV-2 Omicron BA.1 variant to ACE2.

[0133] Please see Figure 32 , Figure 32A bar chart is shown illustrating the binding rates of the four extracts from Example 1 to ACE2 of the Omicron BA.2 variant of SARS-CoV-2. The results showed that the 2 mg / mL crude extract, ethyl acetate extract, n-butanol extract, and aqueous extract all significantly inhibited the binding of the Omicron BA.2 variant of SARS-CoV-2 to ACE2; the aqueous extract also showed a trend towards inhibiting binding.

[0134] Please see Figure 33 , Figure 33 A bar graph illustrating the binding rates of four extracts from some of the disclosed embodiments of the novel coronavirus Omicron BA.4 / 5 variant to ACE2 is presented (the binding sites of BA.4 and BA.5 have the same sequence). The results show that 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 variant to ACE2. Furthermore, the graph shows that the inhibitors were not effective in inhibiting the binding of the novel coronavirus Omicron BA.4 / 5 variant to ACE2.

[0135] Example 5 Neutralization test of extracts and compounds against SARS-CoV-2 For the virus neutralization assay, 1,000 TU of SARS-CoV-2 pseudotyped lentivirus was used to infect 10,000 stable HEK-293T cells expressing the human ACE2 gene in 96-well plates. Simultaneously, different concentrations of extracts or compounds from different species in Example 1 were added, and the binding of the virus to ACE2 was observed, with the IC50 (half-inhibitory concentration) recorded. 50 .

[0136] Please see Figure 34 , Figure 34 The graph illustrates the binding rate of the crude extract of Example 1 to the Omicron BA.2 variant of SARS-CoV-2 and ACE2. The results show that the half-maximal inhibitory concentration (IC50) is [missing value]. 50 It was 77.8 μg / mL.

[0137] Please see Figure 35 , Figure 35 The graph illustrates the binding rate of the crude extract of Example 1 to ACE2 of the Omicron BA.4 / 5 variant of SARS-CoV-2. The results show that the half-maximal inhibitory concentration (IC50) is [missing value]. 50 It was 77.4 μg / mL.

[0138] Please see Figure 36 , Figure 36 The ethyl acetate extract of Example 1 illustrates the binding rate of the SARS-CoV-2 Omicron BA.2 variant to ACE2. The results show that the half-maximal inhibitory concentration (IC50) is [value missing]. 50 It is 74 μg / mL.

[0139] Please see Figure 37 , Figure 37 The ethyl acetate extract of Example 1 illustrates the binding rate of the SARS-CoV-2 Omicron BA.4 / 5 variant to ACE2. The results show that the half-maximal inhibitory concentration (IC50) is [value missing]. 50 It is 100 μg / mL.

[0140] Please see Figures 38A to 38J , Figures 38A to 38J The binding rates of compounds Cd1 to Cd10 from Example 1 to ACE2 of the SARS-CoV-2 Omicron BA.1 variant are illustrated. The results show that the half-maximal inhibitory concentration (IC50) of Cd1 to Cd10 is [missing value]. 50 They are 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.

[0141] Please see Figures 39A to 39J , Figures 39A to 39J The binding rates of compounds Cd1 to Cd10 from Example 1 to ACE2 of the SARS-CoV-2 Omicron BA.2 variant are illustrated. The results show that the half-maximal inhibitory concentration (IC50) of Cd1 to Cd10 is [missing value]. 50 They are 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.

[0142] Please see Figures 40A to 40J , Figures 40A to 40J The binding rates of compounds Cd1 to Cd10 from Example 1 to ACE2 of the SARS-CoV-2 Omicron BA.4 / 5 variant are illustrated. The results show that the half-maximal inhibitory concentration (IC50) of Cd1 to Cd10 is [missing information]. 50 They are 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.

[0143] In some embodiments, the crude extract, ethyl acetate layer extract, n-butanol layer extract, and aqueous layer extract group showed an inhibitory binding effect on the original SARS-CoV-2 strain and ACE2.

[0144] In some embodiments, the crude extract and ethyl acetate extract have an inhibitory binding effect on the A1pha variant of SARS-CoV-2 and ACE2.

[0145] In some embodiments, the crude extract, ethyl acetate layer extract, n-butanol layer extract (greater than 2 mg / mL), and aqueous layer extract (greater than 0.5 mg / mL) have an inhibitory binding effect on the binding of the SARS-CoV-2 Beta variant to ACE2.

[0146] In some embodiments, crude extract, ethyl acetate layer extract, and n-butanol layer extract at concentrations of 1.25–2.5 mg / mL have an inhibitory effect on the binding of the Delta variant of SARS-CoV-2 to ACE2.

[0147] In some embodiments, crude extract, ethyl acetate layer extract, and n-butanol layer extract at concentrations of 1.25–5 mg / mL have an inhibitory binding effect on the RBD region of the Delta variant of SARS-CoV-2 and ACE2.

[0148] In some embodiments, the crude extract, ethyl acetate layer extract, and n-butanol layer extract have an inhibitory binding effect on the binding of SARS-CoV-2 BA.1 and BA.2 variants to ACE2.

[0149] In some embodiments, the crude extract, ethyl acetate layer extract, n-butanol layer extract, and aqueous layer extract have an inhibitory binding effect on the BA.4 / 5 variant of SARS-CoV-2 and ACE2.

[0150] In some embodiments, compounds Cd1 to Cd10 have an inhibitory binding effect on SARS-CoV-2 and ACE2.

[0151] Although the present disclosure has been described above with reference to embodiments, it is not intended to limit the present disclosure. Those skilled in the art can make various modifications and refinements without departing from the spirit and scope of the present disclosure. Therefore, the scope of protection of the present disclosure shall be determined by the appended claims.

Claims

1. A crude citrus extract or extract that inhibits the novel coronavirus, characterized in that, It is prepared by the following steps: The citrus fruit provided is the Taiwanese lemon; The citrus fruit was extracted with ethanol. The citrus fruit was extracted with 95% ethanol at a weight-to-volume ratio of 1:10 to obtain the liquid fraction and the extracted fruit. After partially drying the liquid, a crude extract is obtained, which inhibits the original strain, Alpha variant, Beta variant, Delta variant, or Omicron variant of the novel coronavirus. The crude extract was partitioned with ethyl acetate and water in a 1:1 ratio to obtain an ethyl acetate layer and a highly polar layer. The ethyl acetate layer was dried to obtain an ethyl acetate extract, which inhibited the original strain, Alpha variant, Beta variant, Delta variant, or Omicron variant of the novel coronavirus. as well as The highly polar layer was partitioned with n-butanol and water in a 1:1 ratio to obtain an n-butanol layer and an aqueous layer. The n-butanol layer was dried to obtain a n-butanol extract, which inhibited the original strain or the Omicron variant of the novel coronavirus. The aqueous layer was dried to obtain an aqueous extract, which inhibited the original strain, the Beta variant, or the Omicron variant of the novel coronavirus. The ethyl acetate extract contains 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-tetramethoxyflavonoids, 5-hydroxy-3',4',6,7,8-pentamethoxyflavonoids, 3',4',5,6,7,8-hexamethoxyflavonoids, and 3',4',5,7,8-pentamethoxyflavanone.

2. A composition for inhibiting the novel coronavirus, characterized in that, It comprises the crude extract, the ethyl acetate extract, the n-butanol extract, the aqueous extract, or a combination thereof as described in claim 1.

3. The composition according to claim 2, wherein, It also contains pharmaceutically acceptable carriers.

4. The composition according to claim 2 or 3, wherein the novel coronavirus comprises the original strain, alpha variant, beta variant, gamma variant, delta variant, omicron variant, or a combination thereof.

5. The composition according to claim 4, wherein the omicron variant comprises BA.1 subtype variant, BA.2 subtype variant, BA.3 subtype variant, BA.4 subtype variant, BA.5 subtype variant, or a combination thereof.

6. The use of a composition in the preparation of a medicament for inhibiting the novel coronavirus, characterized in that, This composition comprises a crude extract, an ethyl acetate extract, a n-butanol extract, or an aqueous extract, and is prepared by the following steps: The citrus fruit provided is the Taiwanese lemon; The citrus fruit was extracted with ethanol. The citrus fruit was extracted with 95% ethanol at a weight-to-volume ratio of 1:10 to obtain the liquid fraction and the extracted fruit. After partially drying the liquid, a crude extract is obtained, which inhibits the original strain, Alpha variant, Beta variant, Delta variant, or Omicron variant of the novel coronavirus. The crude extract was partitioned with ethyl acetate and water in a 1:1 ratio to obtain an ethyl acetate layer and a highly polar layer. The ethyl acetate layer was dried to obtain an ethyl acetate extract, which inhibited the original strain, Alpha variant, Beta variant, Delta variant, or Omicron variant of the novel coronavirus. as well as The highly polar layer was partitioned with n-butanol and water in a 1:1 ratio to obtain an n-butanol layer and an aqueous layer. The n-butanol layer was dried to obtain a n-butanol extract, which inhibited the original strain or the Omicron variant of the novel coronavirus. The aqueous layer was dried to obtain an aqueous extract, which inhibited the original strain, the Beta variant, or the Omicron variant of the novel coronavirus.

7. The use according to claim 6, wherein, It also contains pharmaceutically acceptable carriers.

8. The use according to claim 6, wherein, The ethyl acetate extract contains 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, 2'-hydroxy-3,3',4,4',5',6'-hexamethoxychalcone, 5-hydroxy-3',4',7,8-tetramethoxyflavonoids, 5-hydroxy-3',4',6,7,8-pentamethoxyflavonoids, 3',4',5,6,7,8-hexamethoxyflavonoids, and 3',4',5,7,8-pentamethoxyflavanone.

9. The use according to claim 6, wherein the omicron variant comprises BA.1 subtype variant, BA.2 subtype variant, BA.3 subtype variant, BA.4 subtype variant, BA.5 subtype variant, or a combination thereof.

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