DESCRIPTION

METHODS TO USE FLAVONOIDS FOR INHIBITION OF SARS-COV-2 AND TREATMENT OF COVID-19

RELATED APPLICATIONS

The presently disclosed subject matter claims the benefit of U.S. Provisional Patent Application Serial No. 63/065,952 filed August 14, 2020 and U.S. Provisional Patent Application Serial No. 63/176,778 filed April 19, 2021, the disclosure of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The presently disclosed subject matter relates in some embodiments to methods and compositions for treating viral infections, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infections by any strain or variant of SARS-CoV-2

ABBREVIATIONS degrees Celsius percent pl microliter μM micromolar

AF (+)-afzelechin

ANR anthocyanidin reductase

ANS anthocyanidin synthase

BSL-3 biosafety level 3

CA (+)-catechin

COVID-19 coronavirus disease 2019

CTE cytopathic effect

Da Dalton

DFR dihydroflavonol reductase

DHK (+)-dihydrokaempferol

DHM (+)-dihydroquercetin

DHQ (+)-dihydroquercetin

DMSO dimethyl sulfoxide

EAF (-)-epiafzelechin EBS ebselen

EC or EPC (-)-epicatechin

EC-DG (-)-epicatechin-3, 5 -digallate

EGC (-)-epigallocatechin

EGC-DG (-)-epigallocatechin-3,5-digallate

EGCG (-)-epigallocatechin-3-O-gallate

GC (+)-gallocatechin

GC-DG gallocatechin-3, 5 -digallate

GCG (-)-gallocatechin gallate hACE2 human angiotensin-converting enzyme 2

HIV Human Immune Deficiency virus

HPLC high performance liquid chromatography

HPV human papillomavirus

HSV Herpes Simplex viruses

IC50 half maximal inhibitory concentration

INN cinanserin

Iso isoquercitrin

Ka kaempferol kg kilogram

LOP lopinavir

M molar

MERS Middle East Respiratory Syndromes mg milligram min minutes ml milliliter mM millimolar

M ^pro main protease

My myricetin ng nanogram nm nanometer

NSP non-structural protein

ORF open reading frame

PA proanthocyandins PA2 = procyanidin A2

PB2 = procyanidin B2

PP = polyprotein

Qu = quercetin

RdRp = RNA-dependent RNA polymerase

RNA = ribonucleic acid

S = spike protein

SARS = severe acute respiratory syndromes

SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2

TLC = thin layer chromatography

UDP = urdine diphosphate

BACKGROUND

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) was first reported to cause a severe pneumonia in December 2019 in Wuhan, China (Wang et al., 2020a; Zhu et al., 2020a; Ding et al., 2020). On February 11, 2020 the World Heath Organization (WHO) designated this pneumonia as coronavirus disease 2019 (COVID-19). COVID-19 then rapidly spread to different countries. On March 11, 2020, WHO announced the COVID- 19 pandemic (WHO, 2020a; WHO, 2020b). Based on the COVID- 19 Dashboard by Center for Systems Science and Engineering at Johns Hopkins Coronavirus Resource Center, by February 7, 2021 106,136,103 cases of infection and 2,316,702 deaths have been reported from more than 200 countries or regions.

The transmission of this contagious virus is complicated. Studies have shown that the virus can be transmitted through gastrointestinal infection (Xiao et al., 2020; Larners et al., 2020) and can stably stay for three hours in air and 72 hours on plastic and steel surfaces (van Doremalen et al., 2020). In addition to causing lung diseases, this virus has also been found to cause other health complications, such as abdominal pain (Larners et al., 2020) and neurologic abnormality (Helms et al., 2020). Surprisingly, a more recent study also revealed that SARS-CoV-2 existed in wastewater from March 2019 (Chavarria-Miro et al., 2020), nine months earlier than the first report from Wuhan. A strategy to stop the spread of the virus was not available until January 2021, when several vaccines started to become available for vaccinations in several countries (Kim et al., 2021; Knoll et al., 2021; Painter et al., 2021; Dooling et al., 2021; CDC, 2021; Gharpure et al., 2021). While infections cases have slowed down since the start of vaccinations, the use of vaccines has also indicated that developing effective medicines is desirable to stop COVID- 19. A recent study showed that mutations of nucleotides encoding new variants of the spike protein of SARS-CoV-2 can cause escape from antibodies (McCarthy et al., 2020). For example, SARS-CoV-2 variant B.1.351 found in South Africa was reported to be able to neutralize vaccines developed by AstraZeneca, Johnson & Johnson (J&J), and Novavax (Cohen, 2021). Merck & Co. stopped their vaccine development to focus instead on developing medicines (Kenilworth, 2021). Unfortunately, to date, effective medicines are still under screening. Although chloroquine and hydroxychloroquine have been reported to be potentially effective in help improve COVID-19 (Wang et al., 2020b) the use of these two anti-malarial medicines is not without potential risk concerns (Bull-Otterson et al., 2020). In June 2020, dexamethasone, a steroid, was reported to decrease death risk in COVID-19 patients (Selvaraj et al., 2020; Khan and Htar, 2020), but more trials are needed to demonstrate its effectiveness. Other potential candidate medicines include the combination of a-interferon and anti-HIV drugs lopinavir/ritonavir (Cao et al., 2020), and remdesivir (Wang et al., 2020b; Holshue et al., 2020). However, again, further studies are still needed to determine if these medicines are effective in treating COVID- 19.

Accordingly, there is an ongoing need for additional methods and compositions for treating COVID-19 and the SARS-CoV-2 infection that causes COVID-19.

SUMMARY

This summary lists several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently disclosed subject matter, whether listed in this summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.

In some embodiments, the presently disclosed subject matter provides a composition for treating a viral infection in a subject in need thereof, the composition comprising an effective amount of a composition selected from the group comprising a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)- epicatechin-3-O-gallate, (±)-epigallocatechin-3-O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, rutin, and any combination thereof; and a carrier. In some embodiments, the composition comprises an extract from green tea.

In some embodiments, the composition comprises a combination of any two compositions independently selected from the group comprising a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)-epicatechin-3-O-gallate, (±)-epigallocatechin-3-O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, and rutin. In some embodiments, a first of the two compositions and a second of the two compositions are present in a ratio ranging from 1:99% by weight to 99:1% by weight of the first to the second composition. In some embodiments, the composition comprises a combination selected from the group comprising: (a) (±)-epigallocatechin-3-O-gallate and quercetin; (b) (±)-epicatechin-3-O-gallate and quercetin; (c) (±)-epigallocatechin-3-O-gallate and isoquercitrin; (d) (±)-epicatechin-3-O-gallate and isoquercitrin; (e) (±)-epigallocatechin- 3-O-gallate and rutin; (f) (±)-epicatechin-3-O-gallate and rutin; (g) procyanidin B1 and quercetin; (h) procyanidin B2 and quercetin; (i) procyanidin B1 and isoquercitrin; (j) procyanidin B2 and isoquercitrin; (k) procyanidin B1 and rutin; and (1) procyanidin B2 and rutin.

In some embodiments, the effective amount is at least about 150 milligrams (mg), optionally wherein the effective amount ranges from about 150 mg to about 500 mg. In some embodiments, at least two compositions selected from the group comprising a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)- epicatechin-3-O-gallate, (±)-epigallocatechin-3-O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, and rutin, and a carrier are present and the effective amount is at least about 150 milligrams (mg) in total of the two compositions, optionally wherein the effective amount ranges from about 150 mg to about 500 mg in total of the two compositions.

In some embodiments, the composition is formulated as a capsule, a tablet, a pill, a sachet, or a drink.

In some embodiments, the composition comprises a molecule produced by metabolic engineering. In some embodiments, metabolic engineering comprises an approach selected from the group comprising a DFR-ANS/LDOX-ANR pathway, a DFR-LAR-ANS/LDOX- ANR pathway, a DFR-LAR pathway, UDP-flavan-3-ol glycosyltransferases, and/or UDP- flavan-3-ol acyltransferase in E. coli, yeast, any tobacco species, and/or plant cells.

In some embodiments, the viral infection is an infection by a virus selected from the group comprising a coronavirus, a Herpes Simplex viruses (HSV), Human Immune Deficiency virus (HIV), human papillomavirus (HPV), a norovirus, a rhinovirus, Zika virus, an ebolavirus, and an influenza virus. In some embodiments, the viral infection is a Severe Acute Respiratory Syndrome (SARS-CoV-2) infection causing coronavirus 2019 (COVID-19). In some embodiments, the effective amount of the composition is effective to inhibit a main protease ( M ^pro ) activity of SARS-CoV-2. In some embodiments, the effective amount of the composition is effective to inhibit human angiotensin-converting enzyme 2 (hACE2) and SARS-CoV-2 spike (S) protein binding in a subject, wherein a SARS-CoV-2 virus is causing COVID-19 in the subject.

In some embodiments, the presently disclosed subject matter provides a method of treating a viral infection in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising an effective amount of a composition selected from the group comprising a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)-epicatechin-3-O-gallate, (±)- epigallocatechin-3-O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, rutin, and any combination thereof; and a carrier.

In some embodiments, the presently disclosed subject matter provides a method of inhibiting M ^pro of SARS-CoV-2, the method comprising contacting the M ^pro of SARS-CoV-2 with an effective amount of a composition comprising an effective amount of a composition selected from the group comprising a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)-epicatechin-3-O-gallate, (±)-epigallocatechin-3- O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, rutin, and any combination thereof; and a carrier.

In some embodiments, the presently disclosed subject matter provides a method of inhibiting hACE2 and SARS-CoV-2 S protein binding in a subject, wherein a SARS-CoV-2 virus is causing COVID-19 in the subject, the method administering to the subject an effective amount of a composition comprising an effective amount of a composition selected from the group comprising a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)-epicatechin-3-O-gallate, (±)-epigallocatechin-3-O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, rutin, and any combination thereof; and a carrier.

In some embodiments, the presently disclosed subject matter provides a method of treating a viral infection in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising a combination selected from the group comprising: (a) (±)-epigallocatechin-3-O-gallate and quercetin; (b) (±)-epicatechin-3- O-gallate and quercetin; (c) (±)-epigallocatechin-3-O-gallate and isoquercitrin; (d) (±)- epicatechin-3-O-gallate and isoquercitrin; (e) (±)-epigallocatechin-3-O-gallate and rutin; (f) (±)-epicatechin-3-O-gallate and rutin; (g) procyanidin B1 and quercetin; (h) procyanidin B2 and quercetin; (i) procyanidin B1 and isoquercitrin; (j) procyanidin B2 and isoquercitrin; (k) procyanidin B1 and rutin; and (1) procyanidin B2 and rutin.

In some embodiments, the presently disclosed subject matter provides a method of inhibiting M ^p '° of SARS-CoV-2, the method comprising contacting the M ^p '° of SARS-CoV-2 with an effective amount of a composition comprising a combination selected from the group comprising: (a) (±)-epigallocatechin-3-O-gallate and quercetin; (b) (±)-epicatechin-3-O- gallate and quercetin; (c) (±)-epigallocatechin-3-O-gallate and isoquercitrin; (d) (±)- epicatechin-3-O-gallate and isoquercitrin; (e) (±)-epigallocatechin-3-O-gallate and rutin; (f) (±)-epicatechin-3-O-gallate and rutin; (g) procyanidin B1 and quercetin; (h) procyanidin B2 and quercetin; (i) procyanidin B1 and isoquercitrin; (j) procyanidin B2 and isoquercitrin; (k) procyanidin B1 and rutin; and (1) procyanidin B2 and rutin.

In some embodiments, the presently disclosed subject matter provides a method of inhibiting hACE2 and SARS-CoV-2 S protein binding in a subject, wherein a SARS-CoV-2 virus is causing COVID- 19 in the subject, the method administering to the subject an effective amount of a composition comprising a combination selected from the group comprising: (a) (±)-epigallocatechin-3-O-gallate and quercetin; (b) (±)-epicatechin-3-O-gallate, and quercetin; (c) (±)-epigallocatechin-3-O-gallate and isoquercitrin; (d) (±)-epicatechin-3-O- gallate and isoquercitrin; (e) (±)-epigallocatechin-3-O-gallate and rutin; (f) (±)-epicatechin-3- O-gallate and rutin; (g) procyanidin B1 and quercetin; (h) procyanidin B2 and quercetin; (i) procyanidin B1 and isoquercitrin; (j) procyanidin B2 and isoquercitrin; (k) procyanidin B 1 and rutin; and (1) procyanidin B2 and rutin

Accordingly, it is an object of the presently disclosed subject matter to provide methods of treating a viral infection, of inhibiting SARS-CoV-2 main protease, of inhibiting hACE2 and SARS-CoV-2 S protein binding, and related compositions.

An object of the presently disclosed subject matter having been stated hereinabove, and which is achieved in whole or in part by the presently disclosed subject matter, other objects will become evident as the description proceeds herein below. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram showing the function of the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) main protease in the virus replication in the host cells. Once the virus enters into the host cells. Its positive sense and single stranded RNA uses the ribosomes to translate open reading frames la and lb to polyproteins (PP), in which the main protease and papain-like protease cleaves PPs to non-structural proteins (NSPs). Three NSPs, RNA dependent RNA polymerase (RdRp), RNA helicase, and exoribonuclease, are involved in the transcription of the positive RNA to negative sense and single stranded RNA, which is further transcribed to its positive sense and single stranded RNA. Finally, structural proteins and a positive single stranded RNA assembly together to form a virus progeny.

Figure 2 is a schematic drawing showing the chemical structures of 12 flavan-3-ol aglycones (labeled with Arabic numerals 1-12) and 48 flavan-3-ol derivatives (13-48) (including flavan-3-O-gallate, flavan-3,5-O-digallates, flavan-3-O-gly cosides, and flavan-3,5- diglycosides). Examples of aglycones includes (+)-afzelechin (1), (-)-epiafzelechin (4), (+)- catechin (2), (-)-epicatechin (5), (+)-gallocatechin (3), and (-)-epigallocatechin (6). Examples of derivatives include (-)-catechin-3-O-gallate (38), (-)-epicatechin-3-O-gallate (17), (-)- gallocatechin-3-O-gallate (45), and (-)-epigallocatechin-3-O-gallate (18), and (-)- epigallocatechin-3, 5-O-digallate (36).

Figure 3 is a schematic drawing showing the chemical structures of eight B-type procyanidin dimers (61-68, e.g. procyanidin B1 and B2) and two A-type procyanidin dimers, Al (69) and A2 (70).

Figures 4A-4H are a series of protein-ligand docking model drawings showing the binding of flavan-3-ols, procyanidin B2, and three potential anti-Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) drugs to the main protease (M ^pro ). Figure 4A is a drawing of the three-dimensional (3D) surface view of the SARS-CoV-2 M ^pro without and with the inhibitor peptide N3. The rectangle frame shows the M ^pro substrate-binding pocket and the darker shaded amino acid residues show the binding of N3 to the M ^pro substratebinding pocket. Figures 4B-4F show models of 10 compounds binding to the M ^pro substratebinding pocket, (+)-afzelechin (AF) and (-)-epiafzelechin (EAF) (Figure 4B); (+)-catechin (CA) and (-)-epicatechin (EPC) (Figure 4C); (+)-gallocatechin (GC) and (-)-epigallocatechin (EGC) (Figure 4D); (-)-gallocatechin gallate (GCG) and (-)-epigallocatechin gallate (EGCG) (Figure 4E); and procyanidin B2 and A2 (Figure 4F). Figure 4G is a drawing of the model of ebselen (EBS) and cinanserin (INN) binding to the M ^pro substratea -binding pocket. Figure 4H is a drawing of the model of lopinavir (LOP) binding to a different position of M ^pro . Figure 5 is a pair of protein-ligand docking model drawings showing the binding of (- )-epicatechin 3, 5-O-digallate (EPC-DG; left) and (-)-epigallocatechin 3, 5-O-digallate (EGC- DG, right) to the main protease (M ^pro ).

Figures 6A-6F are a series of drawings showing the binding of ebselen, (-)- epigallocatechin (EGC), (-)-epigallocatechin-3-O-gallate (EGCG), (-)-epigallocatechin-3,5- O-digallate (EGC-DG), procyanidin A2, and procyanidin B2 to subsites of main protease (MP™) predicted by protein-ligand docking modeling and the prediction of hydrogen bounds. Figure 6A is a drawing showing ebselen binding to the S1 and S1’ subsites M ^pro . Figure 6B is a drawing showing EGC binding to the S1, S2/S4 subsites of M ^pro . Figure 6C is a drawing showing EGCG binding to the S1, S1’, and S2 subsites of M ^pro . Figure 6D is a drawing showing EGC-DG binding to the S1, S1’, S2 and S4 subsites of M ^pro . Figure 6E is a drawing showing procyanidin Al binding to the S1’, S1, and S4 subsites of M ^pro . Figure 6F is a drawing showing procyanidin B2 binding to the S1, S1’, and S2 subsites of M ^pro .

Figures 7A-7E are a series of schematic drawings of hydrogen bonds predicted by modeling showing different numbers and linkage positions between main protease (M ^pro ) and six flavan-3-ol aglycones, gallocatechin gallate (GCG), epigallocatechin gallate (EGCG), procyanidin A2 or procyanidin B2. Figure 7A is a drawing showing predicted hydrogen bonds for (+)-afzelechin and (-)-epiafzelechin with M ^pro Figure 7B is a drawing showing predicted hydrogen bonds for (+)-catechin and (-)-epicatechin with M ^pro . Figure 7C is a drawing showing predicted hydrogen bonds for (+)-gallocatechin and (-)-epigallocatechin with M ^pro Figure 7D is a drawing showing predicted hydrogen bonds for (+)-gallocatechin gallate and (- )-epigallocatechin gallate with M ^pro . Figure 7E is a drawing showing predicted hydrogen bonds for procyanidin A2 and procyanidin B2 with M ^pro

Figures 8A-8G are a series of graphs showing the inhibitory effects of three flavan-3- ol-gallates, procyanidin A2 (PA2), and procyanidin B2 (PB2) on the main protease (M ^pro ) activity of Severe Acute Respiratory coronavirus 2 (SARS-CoV-2). Figures 8A-8D and Figure 8G are graphs of dynamic curves showing that (+)-catechin-3-O-gallate (CAG) (Figure 8 A), (-)-gallocatechin-3-O-gallate (GCG) (Figure 8B), (-)-epigallocatechin-3-O-gallate (EGCG) (Figure 8C), PB2 (Figure 8D), and (-)-epicatechin-3-O-gallate (ECG) (Figure 8G) inhibit the activity of M ^p '° with an half maximal inhibitory concentration (IC ₅₀ ) of 2.98±0.21 micromolar (μM), 6.38±0.5 μM, 7.51±0.21 μM, 75.31±1.29 μM, and 5.21 μM, respectively. Figure 8E is a graph showing eight concentrations (0-200 μM) of (-)-epigallocatechin (EGC) tested do not show inhibition on the activity of M ^pro . Figure 8F is a graph of the inhibitory effects of 100 μM (-)-epicatechin (EP), (+)-catechin (CA), EGC, CAG, ECG, GCG, EGCG, PA2, and PB2 on the activity of M ^pro . Compared with the negative control, the activity of M ^pro is reduced by 40%, 40%, 44.4%, 50%, 81.5%, and 100% by 100 μM CAG, ECG, EGCG, GCG, PB2, and positive control GC376.

Figures 9A-9D are a series of graphs showing that there are no inhibitory effects of (- )-epicatechin (EP, Figure 9A), (+)-catechin (CA, Figure 9B), (-)-gallocatechin (GC, Figure 9C), and procyanidin A2 (PA2, Figure 9D) on the activity of the main protease (M ^pro ).

Figures 10A-10F are a series of graphs showing the inhibitory effects of five crude extracts on main protease (M ^pro ) activity. Figures 10A-10E are graphs of dynamic curves showing inhibitory activities of different concentrations of extracts from green tea with half maximal inhibitory concentrations (IC ₅₀ ) of 2.84 ±0.25 micrograms per milliliter (pg/ml) (Figure 10A), FLH13-11 muscadine berry IC ₅₀ 29.54 ±0.41 pg/ml (Figure 10B), FLH17-66 berry with IC ₅₀ 29.93 ±0.83 pg/ml (Figure 10C), cacao with IC ₅₀ 153.3 ±47.3 pg/ml (Figure 10D), and dark chocolate IC ₅₀ 256.39 ±66.3 pg/ml (Figure 10E). Figure 10F is a graph showing the inhibition of M ^pro activity by 100 pg/ml extracts of green tea, cacao, chocolate, FLH13-11 muscadine berry, and FLH17-66 muscadine berry. GC376 (100 pg/ml) and 1% DMSO in water were used as positive and negative controls. All values are averaged from five replicates.

Figures 11 A-l ID are schematic drawings of protein-ligand docking modeling between four compounds and human angiotensin-converting enzyme 2 (hACE2) and Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein. Docking modeling shows that (-)-epigallocatechin (EGC) (Figure 11 A), (-)-epigallocatechin-3-O-gallate (EGCG) (Figure 11B), (-)-epicatechin-3,5-O-digallate (EPC-DG) (Figure 11C), and (-)- epigallocatechin-3,5-O-digallate (EGC-DG) (Figure 11D) can dock between the interacting space of hACE2 and S proteins.

Figure 12 is a schematic drawing of the chemical structures of ebselen and 10 flavonoids. Two flavan-3-ols: (-)-epicatechin and (±)-catechin; three dihydroflavonol aglycones: (±)-dihydroquercetin, (±)-dihydrokaempferol, and (±)-dihydroquercetin; three flavonol aglycones, kaempferol, quercetin, and myricetin; two glycosylated flavonols: quercetin-3-O-gly coside (isoquercitrin), and rutin.

Figure 13 is a series of schematic drawings of ligand-receptor docking models showing the binding of eleven compounds to the substrate pocket of the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) main protease (M ^pro ). The first image (top left) shows the three-dimensional (3D) surface view of the SARS-CoV-2 M ^pro , on which the rectangular frame indicates the substrate-binding pocket. Eleven flavonoids and ebselen bind to this pocket. Two flavan-3-ols: (±)-catechin (CA) and (-)-epicatechin (EC); three dihydroflavonol aglycones: (±)-dihydroquercetin (DHQ), (±)-dihydrokaempferol (DHK), and (±)- dihydroquercetin (DHM); three flavonol aglycones, kaempferol, quercetin, and myricetin; two glycosylated flavonols: quercetin-3-O-gly coside (isoquercitrin), and rutin.

Figures 14A-14J are a series of schematic drawings showing the orientation features of compounds binding to subsites. Figure 14A is a schematic drawing that shows a schematic surface image of the four subsites in the binding pocket. Figures 14B-14J are schematic drawings that show images of the binding positions of nine compounds: ebselen (Figure 14B); three dihydroflavonol aglycones, i.e., (+)-dihydroquercetin (DHQ) (Figure 14D), (+)- dihydrokaempferol (DHK) (Figure 14C), and (+)-dihydroquercetin (DHM) (Figure 14E); three flavonol aglycones, i.e., kaempferol (Figure 14F), quercetin (Figure 14G), and myricetin (Figure 14H); and two glycosylated flavonols:, i.e., quercetin-3-O-gly coside (isoquercitrin) (Figure 141), and rutin (Figure 14 J).

Figures 15A-15H are a series of graphs showing the inhibitory effects of nine compounds on the activity of the main protease ( M ^pro ) of Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2). Figures 15A-15G are a series of seven plots showing the inhibitory curves of seven compounds (kaempferol, Figure 15 A; quercetin, Figure 15B; myricetin, Figure 15C; taxifolin, Figure 15D; (+)-dihydroquercetin (DHM), Figure 15E; isoquercitrin, Figure 15F; and rutin, Figure 15G) against the M ^pro activity. All dots in each plot are an average value calculated from five replicates. A half maximal inhibitory activity (IC50) value for each compound is inserted in each plot. “95% C1” means 95% confidence internal, “(value 1, value 2)” means values in the range with 95% C1. Figure 15H is a graph showing a comparison of the inhibitory effects of 11 compounds at 100 μM on the M ^pro activity. GC376 is an inhibitor used as positive control. (+)-catechin, (-)-epicatechin, and water are used as negative controls. Two flavan-3-ols: (+)-catechin (Ca) and (-)-epicatechin (Ep); three dihydroflavonol aglycones: (+)-dihydroquercetin (DHQ), (+)-dihydrokaempferol (DHK), and (+)-dihydroquercetin (DHM); three flavonol aglycones, kaempferol (Ka), quercetin (Qu), and myricetin (My); two glycosylated flavonols: quercetin-3-O-glycoside (isoquercitrin, Iso), and rutin.

Figures 16A and 16B are graphs showing the inhibitory effect of quercetin on the replication of the 229E strain of human coronavirus (HCoV-229E) in human hepatoma- derived Huh-7 cells. Figure 16A is a graph showing the inhibition of HCoV-229E in Huh-7 cells by different concentrations of quercetin (0 micromolar (μM), 2.5 μM, 5 μM, 10 μM, 20 μM or 50 μM). Bars labeled with mean there was a significant difference compared with control (P- value less than 0.05). Figure 16B is a graph showing the inhibition rate (in percentage (%)) versus the log of quercetin concentration (in μM) used to estimate the half maximal effective concentration (EC50) of quercetin as 4.88 μM. “95% Cl” means 95% confidence interval, “(value 1, value 2)” means values in the range with 95% Cl.

Figure 17 is a graph showing the inhibitory effect of quercetin-3-O-gly coside (isoquercitrin) on the replication of the 229E strain of human coronavirus (HCoV-229E) in human hepatoma-derived Huh-7 cells. The graph shows inhibitory effect of different concentrations of isoquercitrin (0 micromolar (μM), 2.5 μM, 5 μM, 10 μM, 20 μM, or 50 μM). Bars labeled with mean there was a significant difference compared with control (P-value less than 0.05).

Figure 18 is a graph showing the inhibitory effect of green tea extract on the replication of the 229E strain of human coronavirus (HCoV-229E) in human hepatoma-derived Huh-7 cells. The graph shows inhibitory effect on the HCoV-229E replication by different amounts of green tea extract (0 micrograms (pg), 2.5 pg, 5 pg, 10 pg, or 20 pg).

Figure 19 is a graph showing the inhibitory effect of (-)-epigallocatechin-3-O-gallate (EGCG) on the replication of the 229E strain of human coronavirus (HCoV-229E) in human hepatoma-derived Huh-7 cells. The graph shows the inhibitory effect on the HCoV-229E replication by different concentrations of EGCG (0 micromolar (μM), 2.5 μM, 5 μM, 10 μM, 20 μM, or 50 μM). Bars labeled with mean there was a significant difference compared with control (P-value less than 0.05).

Figure 20 is a graph showing the inhibitory effect of epicatechin on the replication of the 229E strain of human coronavirus (HCoV-229E) in human hepatoma-derived Huh-7 cells. The graph shows the inhibitory effect of different concentrations of epicatechin (0 micromolar (μM), 2.5 μM, 5 μM, 10 μM, 20 μM, or 50 μM). Bars labeled with mean there was a significant difference compared with control (P-value less than 0.05).

Figure 21 is a graph showing the inhibitory effect of taxifolin (dihydroquercetin) on the replication of the 229E strain of human coronavirus (HCoV0229E) in human hepatoma- derived Huh-7 cells. The graph shows the inhibitory effect of different concentrations of taxifolin (0 micromolar (μM), 2.5 μM, 5 μM, 10 μM, 20 μM, or 50 μM). Bars labeled with mean there was a significant difference compared with control (P-value less than 0.05).

DETAILED DESCRIPTION

The presently disclosed subject matter will now be described more fully. The presently disclosed subject matter can, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein below and in the accompanying Examples. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the embodiments to those skilled in the art. All references listed herein, including but not limited to all patents, patent applications and publications thereof, and scientific journal articles, are incorporated herein by reference in their entireties to the extent that they supplement, explain, provide a background for, or teach methodology, techniques, and/or compositions employed herein.

I, Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this presently described subject matter belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently claimed subject matter.

Following long-standing patent law convention, the terms "a", "an", and “the” refer to "one or more" when used herein, including in the claims.

As used herein, the term “about”, when referring to a value or an amount, for example, relative to another measure, is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, and in some embodiments ±0.1% from the specified value or amount, as such variations are appropriate. The term “about” can be applied to all values set forth herein.

As used herein, ranges can be expressed as from “about” one particular value, and/or to “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

As used herein, the term “and/or” when used in the context of a listing of entities, refers to the entities being present singly or in combination. Thus, for example, the phrase “A, B, C, and/or D” includes A, B, C, and D individually, but also includes any and all combinations and sub-combinations of A, B, C, and D.

The term “comprising”, which is synonymous with “including,” “containing,” or “characterized by” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. “Comprising” is a term of art used in claim language which means that the named elements are present, but other elements can be added and still form a construct or method within the scope of the claim. As used herein, the phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. When the phrase “consists of’ appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

As used herein, the phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.

With respect to the terms “comprising”, “consisting of’, and “consisting essentially of’, where one of these three terms is used herein, the presently disclosed and claimed subject matter can include the use of either of the other two terms.

As used herein, “significance” or “significant” relates to a statistical analysis of the probability that there is a non-random association between two or more entities. To determine whether or not a relationship is “significant” or has “significance”, statistical manipulations of the data can be performed to calculate a probability, expressed in some embodiments as a “p- value”. Those p-values that fall below a user-defined cutoff point are regarded as significant. In some embodiments, a p-value less than or equal to 0.05, in some embodiments less than 0.01, in some embodiments less than 0.005, and in some embodiments less than 0.001, are regarded as significant.

“Concurrently administering” as used herein refers to the administration of two or more separate compounds or compositions in close temporal proximity to one another (for example, simultaneously or sequentially). Concurrent administration may optionally be carried out by administering the two or more compounds or compositions together in a common carrier.

The term “subject,” as used herein, generally refers to a mammal. Typically, the subject is a human. However, the term embraces other species, e.g., pigs, mice, rats, dogs, cats, or other primates. In certain embodiments, the subject is an experimental subject such as a mouse or rat. The subject may be a male or female. The subject may be an infant, a toddler, a child, a young adult, an adult or a geriatric. A subject under the care of a physician or other health care provider may be referred to as a “patient”.

A “subject” of diagnosis or treatment is an animal, including a human. It also includes pets and livestock.

As used herein, a “subject in need thereof’ is a patient, animal, mammal, or human, who will benefit from the method of the presently disclosed subject matter. “Synergistically” as used herein means that the combined effect of two separate active agents is greater than that which would be expected from the sum of the two agents when administered separately.

As used herein, the term “treating” may include prophylaxis of the specific injury, disease, disorder, or condition, or alleviation of the symptoms associated with a specific injury, disease, disorder, or condition and/or preventing or eliminating said symptoms. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease. “Treating” is used interchangeably with “treatment” herein.

In some embodiments, an effective amount of a composition of the presently disclosed subject matter is administered to a subject in need thereof. A “treatment effective amount” or “effective amount” as those phrases are used herein is an amount of a composition sufficient to produce a measurable response (e.g., a biologically or clinically relevant response in a subject being treated). Actual levels of an active agent or agents in the compositions of the presently disclosed subject matter can be varied so as to administer an amount of the active agent(s) that is effective to achieve the desired response for a particular subject.

The selected treatment effective amount can also depend upon combination with other drugs or treatments, the severity of the condition being treated, and the condition and prior medical history of the subject being treated. However, upon a review of the instant disclosure, it is within the skill of the art to start effective amounts of the compositions of the presently disclosed subject matter at levels lower than required to achieve the desired treatment effect and to gradually increase the effective amount until the desired treatment effect is achieved. The potency of a composition can vary, and therefore an “effective amount” can vary. However, upon review of the instant disclosure one skilled in the art can readily assess the potency and efficacy of a bioactive composition of the presently disclosed subject matter and adjust the treatment regimen accordingly.

As used herein, the term “derivative” refers to a chemical compound that can be produced from another compound of similar structure in one or more chemical or biochemical steps as would be apparent to one of ordinary skill in the art upon a review of the instant disclosure, e.g., via the replacement of a H by an alkyl, aryl, aralkyl, acyl, amino or other chemical group as disclosed herein. In some embodiments, a derivative is prepared using the bonding profiles show in the Figures to modify (e.g., enhance) interactions with one or more amino acids in a target protein. As used herein, an “analog” of a chemical compound is a compound that, by way of example, resembles another in structure but is not necessarily an isomer (e.g., 5 -fluorouracil is an analog of thymine). In some embodiments, an analog is prepared using the bonding profiles show in the Figures to modify (e.g., enhance) interactions with one or more amino acids in a target protein.

As used herein the term “alkyl” refers to C1-20 inclusive, linear (i.e., "straight-chain"), branched, or cyclic, saturated or at least partially and in some cases fully unsaturated (i.e., alkenyl and alkynyl) hydrocarbon chains, including for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups. "Branched" refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain. In some embodiments, the alkyl group is “lower alkyl.” "Lower alkyl" refers to an alkyl group having 1 to about 8 carbon atoms (i.e., a C1-8 alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. In some embodiments, the alkyl is “higher alkyl.” "Higher alkyl" refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments, "alkyl" refers, in particular, to C1-8 straight-chain alkyls. In other embodiments, “alkyl” refers, in particular, to C1-8 branched-chain alkyls.

Alkyl groups can optionally be substituted (a “substituted alkyl”) with one or more alkyl group substituents, which can be the same or different. The term "alkyl group substituent" includes but is not limited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. There can be optionally inserted along the alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, lower alkyl (also referred to herein as “alkylaminoalkyl”), or aryl.

Thus, as used herein, the term "substituted alkyl" includes alkyl groups, as defined herein, in which one or more atoms or functional groups of the alkyl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.

The term "aryl" is used herein to refer to an aromatic moiety that can be a single aromatic ring, or multiple aromatic rings that are fused together, linked covalently, or linked to a common group, such as, but not limited to, a methylene or ethylene moiety. The common linking group also can be a carbonyl, as in benzophenone, or oxygen, as in diphenylether, or nitrogen, as in diphenyl amine. The term "aryl" specifically encompasses heterocyclic aromatic compounds. The aromatic ring(s) can comprise phenyl, naphthyl, biphenyl, diphenyl ether, diphenylamine and benzophenone, among others. In particular embodiments, the term “aryl” means a cyclic aromatic comprising about 5 to about 10 carbon atoms, e.g., 5, 6, 7, 8, 9, or 10 carbon atoms, and including 5- and 6-membered hydrocarbon and heterocyclic aromatic rings.

The aryl group can be optionally substituted (a “substituted aryl”) with one or more aryl group substituents, which can be the same or different, wherein “aryl group substituent” includes alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, aryloxyl, aralkyloxyl, carboxyl, carbonyl, acyl, halo, nitro, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxyl, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio, alkylene, and -NR'R", wherein R' and R" can each be independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and aralkyl.

Thus, as used herein, the term "substituted aryl" includes aryl groups, as defined herein, in which one or more atoms or functional groups of the aryl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.

Specific examples of aryl groups include, but are not limited to, cyclopentadienyl, phenyl, furan, thiophene, pyrrole, pyran, pyridine, imidazole, benzimidazole, isothiazole, isoxazole, pyrazole, pyrazine, triazine, pyrimidine, quinoline, isoquinoline, indole, carbazole, and the like.

The term “heteroaryl” refers to aryl groups wherein at least one atom of the backbone of the aromatic ring or rings is an atom other than carbon. Thus, heteroaryl groups have one or more non-carbon atoms selected from the group including, but not limited to, nitrogen, oxygen, and sulfur.

As used herein, the term "acyl" refers to an organic carboxylic acid group wherein the -OH of the carboxyl group has been replaced with another substituent (i.e., as represented by RCO — , wherein R is an alkyl or an aryl group as defined herein). As such, the term "acyl" specifically includes arylacyl groups, such as an acetylfuran and a phenacyl group. Specific examples of acyl groups include acetyl and benzoyl.

“Cyclic” and "cycloalkyl" refer to a non-aromatic mono- or multicyclic ring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The cycloalkyl group can be optionally partially unsaturated. The cycloalkyl group also can be optionally substituted with an alkyl group substituent as defined herein, oxo, and/or alkylene. There can be optionally inserted along the cyclic alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl, thus providing a heterocyclic group. Representative monocyclic cycloalkyl rings include cyclopentyl, cyclohexyl, and cycloheptyl. Multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl.

The terms “heterocycle” or “heterocyclic” refer to cycloalkyl groups (i.e., nonaromatic, cyclic groups as described hereinabove) wherein one or more of the backbone carbon atoms of a cyclic ring is replaced by a heteroatom (e.g., nitrogen, sulfur, or oxygen). Examples of heterocycles include, but are not limited to, tetrahydrofuran, tetrahydropyran, morpholine, dioxane, piperidine, piperazine, and pyrrolidine.

“Alkylene" refers to a straight or branched bivalent aliphatic hydrocarbon group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The alkylene group can be straight, branched or cyclic. The alkylene group also can be optionally unsaturated and/or substituted with one or more "alkyl group substituents." There can be optionally inserted along the alkylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms (also referred to herein as “alkylaminoalkyl”), wherein the nitrogen substituent is alkyl as previously described. Exemplary alkylene groups include methylene (-CH2-); ethylene (-CH2-CH2-); propylene (- (CH ₂ ) ₃ -); cyclohexylene (-C ₆ H ₁₀ -); -CH=CH— CH=CH-; -CH=CH-CH ₂ -; -(CH ₂ ) _q -N(R)- (CH2) ₁ -, wherein each of q and r is independently an integer from 0 to about 20, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and R is hydrogen or lower alkyl; methylenedioxyl (-O-CH2-O-); and ethyl enedioxyl (-O-(CH2)2-O-). An alkylene group can have about 2 to about 3 carbon atoms and can further have 6-20 carbons.

"Alkoxyl" or “alkoxy” refers to an alky 1-O- group wherein alkyl is as previously described. The term "alkoxyl" as used herein can refer to, for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, butoxyl, /-butoxy I, and pentoxyl. The term “oxyalkyl” can be used interchangably with “alkoxyl”.

"Aralkyl" refers to an aryl-alkyl- group wherein aryl and alkyl are as previously described, and included substituted aryl and substituted alkyl. Exemplary aralkyl groups include benzyl, phenylethyl, and naphthylmethyl.

The term “amino” refers to the -NR’R” group, wherein R’ and R” are each independently selected from the group including H and substituted and unsubstituted alkyl, cycloalkyl, heterocycle, aralkyl, aryl, and heteroaryl. In some embodiments, the amino group is -NH2. The term “carbonyl” refers to the -(C=O)- or a double bonded oxygen substituent attached to a carbon atom of a previously named parent group.

The term “carboxyl” refers to the -COOH group.

The terms "halo", "halide", or "halogen" as used herein refer to fluoro, chloro, bromo, and iodo groups.

The terms "hydroxyl" and “hydroxy” refer to the -OH group.

The term “oxo” refers to a compound described previously herein wherein a carbon atom is replaced by an oxygen atom.

The term “cyano” refers to the -CN group.

The term “nitro” refers to the -NO2 group.

II. General Considerations

SARS-CoV-2 is a single stranded RNA virus. Its genomic RNA contains around 30,000 nucleotides and forms a positive sense strand with a 5' methylated cap and a 3' polyadenylated tail that encodes at least six open reading frames (ORF) (Chen et al., 2020; Hussain et al., 2005). This feature allows it to be able to use the ribosomes of the host cells to translate proteins. The longest ORF (ORFla/b) translates two polyproteins, which are cleaved by one main protease (M ^pro , a 3C-like protease, 3CL ^pro ) and another papain-like protease (PL2pro) into 16 nonstructural proteins (NSPs), which include RNA-dependent RNA polymerase (RdRp, nsp12), RNA helicase (nsp13), and exoribonuclease (nsp14). The NSPs subsequently involve the production of structural and accessary proteins. The structural proteins include an envelope (E) protein, membrane (M) protein, spike (S) protein, and nucleocapsid (N) protein (Ramajayam et al., 2011; Ren et al., 2013). See Figure 1. The S, E, and M proteins form the viral envelope. The N protein embraces the single stranded RNA genome. The S protein forms the viral corona appearance and is responsible for the virus’ attachment to and fusion with host cells via the human angiotension-converting enzyme 2 (ACE2) complex.

Given that not only does the SARS-CoV-2 M ^pro play a vital role in the cleavage of polyproteins, but also that the human genome does not have a homolog, it is a desirable target for anti-SARS-CoV-2 drug screening and development (Yang et al., 2005; and Kim et al., 2016). Its high-resolution crystal structure was elucidated in April 2020 (Jin et al., 2020d). It is a cysteine protease and has a Cys-His catalytic dyad, which is site of interest for design and screening of antiviral drugs (Dai et al., 2020). Based on the crystal structure, screening the existing antiviral medicines or designed chemicals revealed that cinanserin, ebselen, GC376, I la, and 1 lb showed inhibitory effects on the M ^pro activity (Jin et al., 2020d; Dai et al., 2020; Ye et al., 2020; Chen et al., 2005; Zhang et al., 2020b). A common feature is that these molecules are that they deliver a carbonyl group (e.g., an aldehyde group or ketone group) to the thiol of the cysteine 145 residue of M ^pro to form a covalent linkage, thus inhibiting M ^pro activity. The potential application of these molecules is still under study to evaluate their effectiveness and side effects.

Flavan-3-ols and proanthocyanidins (PAs) are two groups of plant flavonoids (Xie and Dixon, 2005). They commonly exist in fruits, food products, and beverages, such as grape (Zhu et al. 2014; Rousserie et al. 2019; lacopini et al. 2008; Yuzuak et al. 2018), strawberry (Fossen et al. 2004; Fischer et al. 2014; Lopez-Serrano and Barcelo 1997), persimmon (Akagi et al. 2009), cranberry (Foo et al. 2000a; Foo et al. 2000b), blueberry (Gu et al. 2002), cacao nuts (Murphy et al. 2003; Miller et al. 2009), chocolate (Schewe et al. 2002; Serafini et al. 2003), green tea (Liu et al. 2012; Zhao et al. 2017; Wang et al. 2020c), and wines (Monagas et al. 2003). Common flavan-3-ol aglycones in these plant products include (-)-epicatechin (EPC), (+)-catechin (CA), (-)-epigallocatechin (EGC), (+)-gallocatechin (GC), (-)- epiafzelechin (EP A), and (+)-afzelechin (AF) (Xie and Dixon 2005). Common flavan-3-ol conjugates include (-)-epicatechin-3-O-gallate (EPCG), (-)-epigallocatechin-3-O-galloate (EGCG), which are highly abundant in green tea (Dai et al. 2020b; Wang et al. 2020c). Chemical structures of exemplary flavan-3-ols are shown in Figure 2.

PAs are oligomeric or polymeric flavan-3-ols. In PAs, the lowest and upper units are called as the starter and extension units, which are linked by interflavan bonds formed between the C8 or C6 of a lower unit and the C4 of an upper unit. In addition, a second linkage is an ether C ₂ -O-C ₇ bond between the starter unit and the extension unit. Based the linkage numbers, PAs are classified into two types of structures, dominant B-type characterized with an interflavan bond only and uncommon A-type featured with an interflavan bond and an ether linkage (Xie and Dixon 2005). Chemical structures of exemplary PAs are shown in Figure 3. Common dimeric B-types in fruits and beverages include procyanidin B 1 , B2, B3 and B4 Two examples of dimeric A-type PAs are procyanidin Al and A2 (Xie and Dixon 2005).

Flavan-3-ols and PAs are potent antioxidants with multiple benefits to human health (Xie and Dixon 2005). Multiple compounds from these two groups, such as CA, EPC, EGC, EGCG, procyanidin B2, and procyanidin A2, have been shown to have antibacterial activity (Molan et al. 2001; Howell et al. 2005), anticancer (Ohata et al. 2005; Suganuma et al. 2011), anti-cardiovascular diseases (Panneerselvam et al. 2010; Loke et al. 2008; MacRae et al. 2019), and anti-aging diseases (Li et al. 2004; Levites et al. 2003; Weinreb et al. 2004).

According to one aspect of the presently disclosed subject matter, to assess whether flavan-3-ol and dimeric PA molecules have anti-SARS-CoV-2 activity, M ^pro docking analysis studies were performed as described hereinbelow. See also Zhu et al., 2020b. Flavan-3-ol gallates, such as (-)-epigallocatechin-3-O-gallate (EGCG), (-)-catechin-3-O-gallate (CAG), and (-)-epicatechin-3 -gallate, and dimeric procyanidins promisingly inhibited the M ^Pro activity. Docking simulation indicated that their inhibitory activity likely resulted from the formation of hydrogen bonds between these compounds and several amino acids in the binding domain of M ^Pro .

Additional groups of plant flavonoids include flavones, flavonols, and dihydroflavonols (Fowler et al., 2009, Hostetler et al., 2017). See Tables 1, 2, and 3, below. See also Figure 12. For example, quercetin is a common flavonol widely existing in plants. As further described herein below, docking simulation was performed for three dihydroflavonols, three flavonols, and two glycosylated quercetins. Then, recombinant M ^Pro was used to test inhibitive activity in vitro. The resulting data showed several compounds effectively inhibited the M ^Pro activity.

The skeleton of flavones and flavonols

Table 1. 75 Flavones and flavonols

The skeleton of flavonol glycosides

Table 2. 11 flavonol glycosides (Glc: glucoside; Rha: rbamnoside; Galgaiactoside; Rob: Robinose ; Rut: Rutinose),

The skeleton of flavanones and flavanonols

Table 3. 16 Flavanones and Flavanonols

III. Methods and Compositions

In some embodiments, the presently disclosed subject matter provides methods and compositions for treating a viral infection in a subject in need thereof. In some embodiments, the presently disclosed subject matter provides a composition for treating a viral infection in a subject in need thereof, wherein the composition comprises an effective amount of a composition selected from the group of compounds or compositions comprising a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)- epicatechin-3-O-gallate, (±)-epigallocatechin-3-O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, rutin, and any combination thereof; and a carrier. By “any combination” it is meant any combination of any two, three, four, or more of the listed compositions. In some embodiments, the composition comprises a composition comprising an extract from green tea. In some embodiments, the composition comprises a combination of any two of the listed compositions, i.e., of any two compounds of compositions independently selected from a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)-epicatechin-3-O-gallate, (±)-epigallocatechin-3- O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, and rutin. In some embodiments, two or more of the compositions provided in combination are chosen on the basis of having similar effectiveness (e.g., similar anti-viral effectiveness).

In some embodiments, the first of the two compositions or compounds and the second of the two compositions or compounds are present in a ratio ranging from about 1:99% by weight to about 99: 1% by weight of the first to the second composition, including about 1 :99% by weight, about 5:95% by weight, about 10:90% by weight, about 20:80% by weight, about 30:70% by weight, about 40:60% by weight, about 50:50% by weight, about 60:40% by weight, about 70:30% by weight, about 80:20% by weight, about 90:10% by weight, about 95:5% by weight, and about 99:1% by weight of the first to the second composition. In the case where more than two component compositions are present, the component compositions can be present in similar proportional ratios.

In some embodiments, the composition comprises a combination of two compounds/compositions selected from the group comprising: (a) (±)-epigallocatechin-3-O- gallate and quercetin; (b) (±)-epicate-chin-3-O-gallate, and quercetin; (c) (±)-epigallo- catechin-3-O-gallate and isoquercitrin; (d) (±)-epicatechin-3-O-gallate and isoquercitrin; (e) (±)-epigallocatechin-3-O-gallate and rutin; (f) (±)-epicatechin-3-O-gallate and rutin; (g) procyanidin B1 and quercetin; (h) procyanidin B2 and quercetin; (i) procyanidin B1 and isoquercitrin; (j) procyanidin B2 and isoquercitrin; (k) procyanidin B1 and rutin; and (1) procyanidin B2 and rutin.

In some embodiments, the effective amount is at least about 150 milligrams (mg). In some embodiments, the effective amount is about 150 mg or about 160 mg. In some embodiments, the effective amount ranges from about 150 mg to about 500 mg, including about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, and about 500 mg. In some embodiments, the effective amount is less than about 150 mg. For example, in some embodiments, the effective amount is about 140 mg, about 130 mg, about 120 mg, about 110 mg, about 100 mg, about 75 mg, about 50 mg, or less. In some embodiments, the effective amount is greater than about 500 mg. Thus, in some embodiments, the effective amount is about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 900 mg, or about 1000 mg or more.

In some embodiments, the composition comprises at least two of the compositions or compounds and the effective amount is at least about 150 milligrams (mg) in total of the two (or three or four or more) listed compositions or compounds, i.e., a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)-epicatechin-3-O- gallate, (±)-epigallocatechin-3-O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, and rutin. Thus, in some embodiments, the composition comprises at least two of the listed compositions and the total weight of the at least two compositions is about 150 mg or at least about 160 mg. In some embodiments, the composition comprises at least two of the listed compositions and the effective amount of the combination of the two or more compositions ranges from about 150 mg to about 500 mg, including about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, and about 500 mg. In some embodiments, a combination comprising three or four compounds, or more, can also be provided, in some embodiments in a total amount of at least about 150 mg, optionally in an amount ranging from about 150 mg to about 500 mg, including about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, and about 500 mg. In some embodiments, the effective amount of the combination of two, three, four, or more compounds is less than about 150 mg (e.g., about 140 mg, about 130 mg, about 120 mg, about 110 mg, about 100 mg, about 75 mg, about 50 mg, or less). In some embodiments, the effective amount of the combination of two, three, four or more compounds is greater than about 500 mg (e.g., about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 900 mg, or about 1000 mg or more). In some embodiments, the effective amount of the single composition or any combination of compositions is sufficient to provide about 2 mg/kg body weight subject/day to about 6.5 mg/kg body weight of subject/day, including about 2 mg/kg body weight subject/day, about 3 mg/kg body weight subject/day, about 4 mg/kg body weight subject/day, about 5 mg/kg body weight subject/day, about 6 mg/kg body weight subject/day about 6.5 mg/kg body weight subject/day.

The composition can be formulated in any suitable form for administration to the subject. In some embodiments, the composition is formulated for oral administration. In some embodiments, the composition is formulated as a capsule, a tablet, a pill, a sachet, or a drink. In some embodiments, the effective amount of the composition or combination of compositions is split between two or more (e.g., three, four, five or more) capsules, tablets, pills, sachets, drinks and/or any other suitable form. In some embodiments, the effective amount is provided in two or more different forms.

In some embodiments, the composition is from a natural source and is provided in the form of a plant extract or a purified component thereof. For convenience, the term “extract” is meant to refer to a plant extract or a purified component thereof. By “purified component thereof’ as used herein refers to a composition that is at least 85% or more (e.g., 90%, 95%, 96%, 97%, 98%, or 99% or more) of a particular component (e.g., a particular flavonoid compound).

Thus, an “extract” as described herein can refer to an extract taken from the leaves, roots, stem, flower, fruits, seeds or any other component of the plant, as appropriate, to provide an extract of interest. If desired, an original extract can be purified to separate individual extracted plant compounds from one another or to enhance the extract for one or more of the individual extracted plant compounds by removing a portion of the other extracted compounds in the original extract. In some embodiments, the term “extract” can refer to compositions obtained by extraction of a named plant, plant part, or powder thereof, with water, an aqueous base, a polar protic organic solvent, a polar aprotic organic solvent, supercritical carbon dioxide, or a mixture thereof. Thus, the extract is a composition comprising one or more compounds (e.g., one or more plant flavonoids) that have been removed from a solid plant, plant part, or plant derived powder via dissolution into a liquid or gas brought into contact with the plant or plant part. The term “extract” includes, but is not limited to, infusions, decoctions, tinctures, syrups, and preparations. In some embodiments, the plant extract composition(s) can be substituted with an active constituent or constituents or with a synthetic equivalent or equivalents that is/are believed to contribute to the improvement or enhancement of the particular composition or formulation thereof. Exemplary one compound formulations and suitable plant sources are described in Table 4, below. Exemplary two compound formulations (with two different exemplary combinations of amounts of the two compounds) are described in Table 5, below. Each exemplary one or two compound formulation can be provided, for example, as a capsule, tablet, pill, sachet, food, or drink. Exemplary plant extract formulations for use according to the presently disclosed subject matter are provided in Table 6, below.

Table 4. Exemplary One Compound Formulations and Plant Sources. Table 5. Exemplary Two Compound Formulations

Table 6. Exemplary Plant Extracts

In some embodiments, the composition comprises a molecule produced by metabolic engineering. The metabolic engineering can comprise an approach selected from the group including, but not limited to, a DFR-ANS/LDOX-ANR, DFR-LAR-ANS/LDOX-ANR, or a DFR-LAR pathway. UDP-flavan-3-ol glycosyltransferases, and/or UDP-flavan-3-ol acyltransferase in E. coli, yeast, any tobacco species, and/or plant cells.

The viral infection can be any viral infection, including both DNA viruses or RNA viruses. In some embodiments, the virus is a virus of the family coronaviridae, herpesviridae, retroviridae, papillomaviridae, caliciviridae, or picomaviridae. In some embodiments, the virus is selected from the group comprising a coronavirus (e.g., SARS, MERS, etc), a Herpes Simplex virus (HSV), Human Immune Deficiency virus (HIV), human papillomavirus (HPV), a norovirus, a rhinovirus, Zika virus, an ebolavirus, and an influenza virus (e.g., influenza type B virus or influenza type C virus). In some embodiments, the virus is a coronavirus. In some embodiments, the virus is SARS-CoV-2, including any strain or variant of SARS-CoV-2. Thus, in some embodiments, the viral infection is a SARS-CoV-2 infection and the subject in need of treatment has COVID- 19.

In some embodiments, the effective amount of the composition is effective to inhibit a main protease (M ^pro ) activity of SARS-CoV-2. In some embodiments, the effective amount of the composition is effective to inhibit human angiotensin-converting enzyme 2 (hACE2) and SARS-CoV-2 spike (S) protein binding in a subject, e.g., wherein a SARS-CoV-2 virus is causing COVID-19 in the subject.

In some embodiments, the presently disclosed subject matter provides a method of treating a viral infection in a subject in need thereof, the method comprising administering to the subject of a composition, wherein the composition comprises an effective amount of a composition selected from the group of compounds or compositions comprising a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)- epicatechin-3-O-gallate, (±)-epigallocatechin-3-O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, rutin, and any combination thereof; and a carrier. By “any combination” it is meant any combination of any two, three, four, or more of the listed compositions. In some embodiments, the composition comprises a composition comprising an extract from green tea. In some embodiments, the composition comprises a combination of any two of the listed compositions, i.e., of any two compounds of compositions independently selected from a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)-epicatechin-3-O-gallate, (±)-epigallocatechin-3- O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, and rutin. In some embodiments, two or more of the compositions provided in combination are chosen on the basis of having similar effectiveness (e.g., similar anti-viral effectiveness).

In some embodiments, the first of the two compositions or compounds and the second of the two compositions or compounds are present in a ratio ranging from about 1:99% by weight to about 99: 1% by weight of the first to the second composition, including about 1 :99% by weight, about 5:95% by weight, about 10:90% by weight, about 20:80% by weight, about 30:70% by weight, about 40:60% by weight, about 50:50% by weight, about 60:40% by weight, about 70:30% by weight, about 80:20% by weight, about 90:10% by weight, about 95:5% by weight, and about 99:1% by weight of the first to the second composition. In the case where more than two component compositions are present, the component compositions can be present in similar proportional ratios.

In some embodiments, the composition comprises a combination of two compounds/compositions selected from the group comprising: (a) (±)-epigallocatechin-3-O- gallate and quercetin; (b) (±)-epicate-chin-3-O-gallate, and quercetin; (c) (±)-epigallo- catechin-3-O-gallate and isoquercitrin; (d) (±)-epicatechin-3-O-gallate and isoquercitrin; (e) (±)-epigallocatechin-3-O-gallate and rutin; (f) (±)-epicatechin-3-O-gallate and rutin; (g) procyanidin B1 and quercetin; (h) procyanidin B2 and quercetin; (i) procyanidin B1 and isoquercitrin; (j) procyanidin B2 and isoquercitrin; (k) procyanidin B1 and rutin; and (1) procyanidin B2 and rutin.

In some embodiments, the effective amount is at least about 150 milligrams (mg). In some embodiments, the effective amount is about 150 mg or about 160 mg. In some embodiments, the effective amount ranges from about 150 mg to about 500 mg, including about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, and about 500 mg. In some embodiments, the composition comprises at least two of the compositions or compounds and the effective amount is at least about 150 milligrams (mg) in total of the two listed compositions or compounds, i.e., a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)-epicatechin-3-O-gallate, (±)-epigallocatechin-3-O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, and rutin. Thus, in some embodiments, the composition comprises at least two of the listed compositions and the total weight of the at least two compositions is about 150 mg or at least about 160 mg. In some embodiments, the composition comprises at least two of the listed compositions and the effective amount of the combination of the two compositions ranges from about 150 mg to about 500 mg, including about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, and about 500 mg. In some embodiments, a combination comprising three or four compounds, or more, can also be provided, in some embodiments in a total amount of at least about 150 mg, optionally in an amount ranging from about 150 mg to about 500 mg, including about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, and about 500 mg. In some embodiments, the effective amount of any single composition or any combination of compositions is less than about 150 mg (e.g, about 140 mg, about 130 mg, about 120 mg, about 110 mg, about 100 mg, about 75 mg, about 50 mg, or less) or greater than about 500 mg (e.g., about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 900 mg, about 1000 mg or more). In some embodiments, the effective amount of the single composition or any combination of compositions is sufficient to provide about 2 mg/kg body weight subject/day to about 6.5 mg/kg body weight of subject/day, including about 2 mg/kg body weight subject/day, about 3 mg/kg body weight subject/day, about 4 mg/kg body weight subject/day, about 5 mg/kg body weight subject/day, about 6 mg/kg body weight subject/day about 6.5 mg/kg body weight subject/day.

In some embodiments, the presently disclosed subject matter provides a method of inhibiting main protease (M ^pro ) of SARS-CoV-2, the method comprising contacting the M ^pro of SARS-CoV-2 with an effective amount of a composition, wherein the composition comprises an effective amount of a composition selected from the group of compounds or compositions comprising a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)-epicatechin-3-O-gallate, (±)-epigallocatechin-3-O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, rutin, and any combination thereof; and a carrier. By “any combination” it is meant any combination of any two, three, four, or more of the listed compositions. In some embodiments, the composition comprises a composition comprising an extract from green tea. In some embodiments, the composition comprises a combination of any two of the listed compositions, i.e., of any two compounds of compositions independently selected from a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)-epicatechin-3-O-gallate, (±)- epigallocatechin-3-O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, and rutin. In some embodiments, two or more of the compositions provided in combination are chosen on the basis of having similar effectiveness (e.g., similar anti-viral effectiveness).

In some embodiments, the first of the two compositions or compounds and the second of the two compositions or compounds are present in a ratio ranging from about 1:99% by weight to about 99: 1% by weight of the first to the second composition, including about 1 :99% by weight, about 5:95% by weight, about 10:90% by weight, about 20:80% by weight, about 30:70% by weight, about 40:60% by weight, about 50:50% by weight, about 60:40% by weight, about 70:30% by weight, about 80:20% by weight, about 90:10% by weight, about 95:5% by weight, and about 99:1% by weight of the first to the second composition. In the case where more than two component compositions are present, the component compositions can be present in similar proportional ratios. In some embodiments, the composition comprises a combination of two compounds/compositions selected from the group comprising: (a) (±)-epigallocatechin-3-O- gallate and quercetin; (b) (±)-epicate-chin-3-O-gallate, and quercetin; (c) (±)-epigallo- catechin-3-O-gallate and isoquercitrin; (d) (±)-epicatechin-3-O-gallate and isoquercitrin; (e) (±)-epigallocatechin-3-O-gallate and rutin; (f) (±)-epicatechin-3-O-gallate and rutin; (g) procyanidin B1 and quercetin; (h) procyanidin B2 and quercetin; (i) procyanidin B1 and isoquercitrin; (j) procyanidin B2 and isoquercitrin; (k) procyanidin B1 and rutin; and (1) procyanidin B2 and rutin.

In some embodiments, the effective amount is at least about 150 milligrams (mg). In some embodiments, the effective amount is about 150 mg or about 160 mg. In some embodiments, the effective amount ranges from about 150 mg to about 500 mg, including about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, and about 500 mg. In some embodiments, the composition comprises at least two of the compositions or compounds and the effective amount is at least about 150 milligrams (mg) in total of the two listed compositions or compounds, i.e., a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)-epicatechin-3-O-gallate, (±)-epigallocatechin-3-O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, and rutin. Thus, in some embodiments, the composition comprises at least two of the listed compositions and the total weight of the at least two compositions is about 150 mg or at least about 160 mg. In some embodiments, the composition comprises at least two of the listed compositions and the effective amount of the combination of the two compositions ranges from about 150 mg to about 500 mg, including about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, and about 500 mg. In some embodiments, a combination comprising three or four compounds, or more, can also be provided, in some embodiments in a total amount of at least about 150 mg, optionally in an amount ranging from about 150 mg to about 500 mg, including about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, and about 500 mg. In some embodiments, the effective amount of any single composition or any combination of compositions is less than about 150 mg (e.g, about 140 mg, about 130 mg, about 120 mg, about 110 mg, about 100 mg, about 75 mg, about 50 mg, or less) or greater than about 500 mg (e.g., about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 900 mg, about 1000 mg or more). In some embodiments, the effective amount of the single composition or any combination of compositions is sufficient to provide about 2 mg/kg body weight subject/day to about 6.5 mg/kg body weight of subject/day, including about 2 mg/kg body weight subject/day, about 3 mg/kg body weight subject/day, about 4 mg/kg body weight subject/day, about 5 mg/kg body weight subject/day, about 6 mg/kg body weight subject/day about 6.5 mg/kg body weight subject/day.

In some embodiments, the presently disclosed subject matter provides a method of inhibiting hACE2 and SARS-CoV-2 S protein binding (e.g., in a subject, wherein a SARS- CoV-2 virus is causing COVID-19 in the subject). In some embodiments, the method comprises contacting the hACE and/or S protein with an effective amount of a composition wherein the composition comprises an effective amount of a composition selected from the group of compounds or compositions comprising a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)-epicatechin-3-O-gallate, (±)- epigallocatechin-3-O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, rutin, and any combination thereof; and a carrier. By “any combination” it is meant any combination of any two, three, four, or more of the listed compositions. In some embodiments, the composition comprises a composition comprising an extract from green tea. In some embodiments, the composition comprises a combination of any two of the listed compositions, i.e., of any two compounds of compositions independently selected from a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)- epicatechin-3-O-gallate, (±)-epigallocatechin-3-O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, and rutin. In some embodiments, two or more of the compositions provided in combination are chosen on the basis of having similar effectiveness (e.g., similar anti-viral effectiveness).

In some embodiments, the first of the two compositions or compounds and the second of the two compositions or compounds are present in a ratio ranging from about 1:99% by weight to about 99: 1% by weight of the first to the second composition, including about 1 :99% by weight, about 5:95% by weight, about 10:90% by weight, about 20:80% by weight, about 30:70% by weight, about 40:60% by weight, about 50:50% by weight, about 60:40% by weight, about 70:30% by weight, about 80:20% by weight, about 90:10% by weight, about 95:5% by weight, and about 99:1% by weight of the first to the second composition. In the case where more than two component compositions are present, the component compositions can be present in similar proportional ratios.

In some embodiments, the composition comprises a combination of two compounds/compositions selected from the group comprising: (a) (±)-epigallocatechin-3-O- gallate and quercetin; (b) (±)-epicate-chin-3-O-gallate, and quercetin; (c) (±)-epigallo- catechin-3-O-gallate and isoquercitrin; (d) (±)-epicatechin-3-O-gallate and isoquercitrin; (e) (±)-epigallocatechin-3-O-gallate and rutin; (f) (±)-epicatechin-3-O-gallate and rutin; (g) procyanidin B1 and quercetin; (h) procyanidin B2 and quercetin; (i) procyanidin B1 and isoquercitrin; (j) procyanidin B2 and isoquercitrin; (k) procyanidin B1 and rutin; and (1) procyanidin B2 and rutin.

In some embodiments, the effective amount is at least about 150 milligrams (mg). In some embodiments, the effective amount is about 150 mg or about 160 mg. In some embodiments, the effective amount ranges from about 150 mg to about 500 mg, including about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, and about 500 mg. In some embodiments, the composition comprises at least two of the compositions or compounds and the effective amount is at least about 150 milligrams (mg) in total of the two listed compositions or compounds, i.e., a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)-epicatechin-3-O-gallate, (±)-epigallocatechin-3-O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, and rutin. Thus, in some embodiments, the composition comprises at least two of the listed compositions and the total weight of the at least two compositions is about 150 mg or at least about 160 mg. In some embodiments, the composition comprises at least two of the listed compositions and the effective amount of the combination of the two compositions ranges from about 150 mg to about 500 mg, including about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, and about 500 mg. In some embodiments, a combination comprising three or four compounds, or more, can also be provided, in some embodiments in a total amount of at least about 150 mg, optionally in an amount ranging from about 150 mg to about 500 mg, including about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, and about 500 mg. In some embodiments, the effective amount of any single composition or any combination of compositions is less than about 150 mg (e.g, about 140 mg, about 130 mg, about 120 mg, about 110 mg, about 100 mg, about 75 mg, about 50 mg, or less) or greater than about 500 mg (e.g., about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 900 mg, about 1000 mg or more). In some embodiments, the effective amount of the single composition or any combination of compositions is sufficient to provide about 2 mg/kg body weight subject/day to about 6.5 mg/kg body weight of subject/day, including about 2 mg/kg body weight subject/day, about 3 mg/kg body weight subject/day, about 4 mg/kg body weight subject/day, about 5 mg/kg body weight subject/day, about 6 mg/kg body weight subject/day about 6.5 mg/kg body weight subject/day. In some embodiments, the presently disclosed subject matter provides a method of treating a viral infection in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition wherein the composition comprises an effective amount of a composition selected from the group of compounds or compositions comprising a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)-epicatechin-3-O-gallate, (±)-epigallocatechin-3-O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, rutin, and any combination thereof; and a carrier. By “any combination” it is meant any combination of any two, three, four, or more of the listed compositions. In some embodiments, the composition comprises a composition comprising an extract from green tea. In some embodiments, the composition comprises a combination of any two of the listed compositions, i.e., of any two compounds of compositions independently selected from a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)-epicatechin-3-O-gallate, (±)-epigallocatechin-3- O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, and rutin. In some embodiments, two or more of the compositions provided in combination are chosen on the basis of having similar effectiveness (e.g., similar anti-viral effectiveness).

In some embodiments, the first of the two compositions or compounds and the second of the two compositions or compounds are present in a ratio ranging from about 1:99% by weight to about 99: 1% by weight of the first to the second composition, including about 1 :99% by weight, about 5:95% by weight, about 10:90% by weight, about 20:80% by weight, about 30:70% by weight, about 40:60% by weight, about 50:50% by weight, about 60:40% by weight, about 70:30% by weight, about 80:20% by weight, about 90:10% by weight, about 95:5% by weight, and about 99:1% by weight of the first to the second composition. In the case where more than two component compositions are present, the component compositions can be present in similar proportional ratios.

In some embodiments, the composition comprises a combination of two compounds/compositions selected from the group comprising: (a) (±)-epigallocatechin-3-O- gallate and quercetin; (b) (±)-epicate-chin-3-O-gallate, and quercetin; (c) (±)-epigallo- catechin-3-O-gallate and isoquercitrin; (d) (±)-epicatechin-3-O-gallate and isoquercitrin; (e) (±)-epigallocatechin-3-O-gallate and rutin; (f) (±)-epicatechin-3-O-gallate and rutin; (g) procyanidin B1 and quercetin; (h) procyanidin B2 and quercetin; (i) procyanidin B1 and isoquercitrin; (j) procyanidin B2 and isoquercitrin; (k) procyanidin B1 and rutin; and (1) procyanidin B2 and rutin. In some embodiments, the effective amount is at least about 150 milligrams (mg). In some embodiments, the effective amount is about 150 mg or about 160 mg. In some embodiments, the effective amount ranges from about 150 mg to about 500 mg, including about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, and about 500 mg. In some embodiments, the composition comprises at least two of the compositions or compounds and the effective amount is at least about 150 milligrams (mg) in total of the two listed compositions or compounds, i.e., a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)-epicatechin-3-O-gallate, (±)-epigallocatechin-3-O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, and rutin. Thus, in some embodiments, the composition comprises at least two of the listed compositions and the total weight of the at least two compositions is about 150 mg or at least about 160 mg. In some embodiments, the composition comprises at least two of the listed compositions and the effective amount of the combination of the two compositions ranges from about 150 mg to about 500 mg, including about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, and about 500 mg. In some embodiments, a combination comprising three or four compounds, or more, can also be provided, in some embodiments in a total amount of at least about 150 mg, optionally in an amount ranging from about 150 mg to about 500 mg, including about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, and about 500 mg. In some embodiments, the effective amount of any single composition or any combination of compositions is less than about 150 mg (e.g, about 140 mg, about 130 mg, about 120 mg, about 110 mg, about 100 mg, about 75 mg, about 50 mg, or less) or greater than about 500 mg (e.g., about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 900 mg, about 1000 mg or more). In some embodiments, the effective amount of the single composition or any combination of compositions is sufficient to provide about 2 mg/kg body weight subject/day to about 6.5 mg/kg body weight of subject/day, including about 2 mg/kg body weight subject/day, about 3 mg/kg body weight subject/day, about 4 mg/kg body weight subject/day, about 5 mg/kg body weight subject/day, about 6 mg/kg body weight subject/day about 6.5 mg/kg body weight subject/day.

In some embodiments, the presently disclosed subject matter provides a method for inhibiting main protease (M ^pro ) of SARS-CoV-2, the method comprising contacting the M ^pro of SARS-CoV-2 with an effective amount of a composition wherein the composition comprises an effective amount of a composition selected from the group of compounds or compositions comprising a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)-epicatechin-3-O-gallate, (±)-epigallocatechin-3-O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, rutin, and any combination thereof; and a carrier. By “any combination” it is meant any combination of any two, three, four, or more of the listed compositions. In some embodiments, the composition comprises a composition comprising an extract from green tea. In some embodiments, the composition comprises a combination of any two of the listed compositions, i.e., of any two compounds of compositions independently selected from a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)-epicatechin-3-O-gallate, (±)- epigallocatechin-3-O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, and rutin. In some embodiments, two or more of the compositions provided in combination are chosen on the basis of having similar effectiveness (e.g., similar anti-viral effectiveness).

In some embodiments, the first of the two compositions or compounds and the second of the two compositions or compounds are present in a ratio ranging from about 1:99% by weight to about 99: 1% by weight of the first to the second composition, including about 1 :99% by weight, about 5:95% by weight, about 10:90% by weight, about 20:80% by weight, about 30:70% by weight, about 40:60% by weight, about 50:50% by weight, about 60:40% by weight, about 70:30% by weight, about 80:20% by weight, about 90:10% by weight, about 95:5% by weight, and about 99: 1% by weight of the first to the second composition. In the case where more than two component compositions are present, the component compositions can be present in similar proportional ratios.

In some embodiments, the composition comprises a combination of two compounds/compositions selected from the group comprising: (a) (±)-epigallocatechin-3-O- gallate and quercetin; (b) (±)-epicate-chin-3-O-gallate, and quercetin; (c) (±)-epigallo- catechin-3-O-gallate and isoquercitrin; (d) (±)-epicatechin-3-O-gallate and isoquercitrin; (e) (±)-epigallocatechin-3-O-gallate and rutin; (f) (±)-epicatechin-3-O-gallate and rutin; (g) procyanidin B1 and quercetin; (h) procyanidin B2 and quercetin; (i) procyanidin B1 and isoquercitrin; (j) procyanidin B2 and isoquercitrin; (k) procyanidin B1 and rutin; and (1) procyanidin B2 and rutin.

In some embodiments, the effective amount is at least about 150 milligrams (mg). In some embodiments, the effective amount is about 150 mg or about 160 mg. In some embodiments, the effective amount ranges from about 150 mg to about 500 mg, including about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, and about 500 mg. In some embodiments, the composition comprises at least two of the compositions or compounds and the effective amount is at least about 150 milligrams (mg) in total of the two listed compositions or compounds, i.e., a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)-epicatechin-3-O-gallate, (±)-epigallocatechin-3-O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, and rutin. Thus, in some embodiments, the composition comprises at least two of the listed compositions and the total weight of the at least two compositions is about 150 mg or at least about 160 mg. In some embodiments, the composition comprises at least two of the listed compositions and the effective amount of the combination of the two compositions ranges from about 150 mg to about 500 mg, including about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, and about 500 mg. In some embodiments, a combination comprising three or four compounds, or more, can also be provided, in some embodiments in a total amount of at least about 150 mg, optionally in an amount ranging from about 150 mg to about 500 mg, including about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, and about 500 mg. In some embodiments, the effective amount of any single composition or any combination of compositions is less than about 150 mg (e.g, about 140 mg, about 130 mg, about 120 mg, about 110 mg, about 100 mg, about 75 mg, about 50 mg, or less) or greater than about 500 mg (e.g., about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 900 mg, about 1000 mg or more). In some embodiments, the effective amount of the single composition or any combination of compositions is sufficient to provide about 2 mg/kg body weight subject/day to about 6.5 mg/kg body weight of subject/day, including about 2 mg/kg body weight subject/day, about 3 mg/kg body weight subject/day, about 4 mg/kg body weight subject/day, about 5 mg/kg body weight subject/day, about 6 mg/kg body weight subject/day about 6.5 mg/kg body weight subject/day.

In some embodiments, the presently disclosed subject matter provides a method of inhibiting hACE2 and SARS-CoV-2 S protein binding in a subject, wherein a SARS-CoV-2 virus is causing COVID- 19 in the subject, the method administering to the subject an effective amount of a composition wherein the composition comprises an effective amount of a composition selected from the group of compounds or compositions comprising a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)- epicatechin-3-O-gallate, (±)-epigallocatechin-3-O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, rutin, and any combination thereof; and a carrier. By “any combination” it is meant any combination of any two, three, four, or more of the listed compositions. In some embodiments, the composition comprises a composition comprising an extract from green tea. In some embodiments, the composition comprises a combination of any two of the listed compositions, i.e., of any two compounds of compositions independently selected from a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)-epicatechin-3-O-gallate, (±)-epigallocatechin-3- O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, and rutin. In some embodiments, two or more of the compositions provided in combination are chosen on the basis of having similar effectiveness (e.g., similar anti-viral effectiveness).

In some embodiments, the first of the two compositions or compounds and the second of the two compositions or compounds are present in a ratio ranging from about 1:99% by weight to about 99: 1% by weight of the first to the second composition, including about 1 :99% by weight, about 5:95% by weight, about 10:90% by weight, about 20:80% by weight, about 30:70% by weight, about 40:60% by weight, about 50:50% by weight, about 60:40% by weight, about 70:30% by weight, about 80:20% by weight, about 90:10% by weight, about 95:5% by weight, and about 99:1% by weight of the first to the second composition. In the case where more than two component compositions are present, the component compositions can be present in similar proportional ratios.

In some embodiments, the composition comprises a combination of two compounds/compositions selected from the group comprising: (a) (±)-epigallocatechin-3-O- gallate and quercetin; (b) (±)-epicate-chin-3-O-gallate, and quercetin; (c) (±)-epigallo- catechin-3-O-gallate and isoquercitrin; (d) (±)-epicatechin-3-O-gallate and isoquercitrin; (e) (±)-epigallocatechin-3-O-gallate and rutin; (f) (±)-epicatechin-3-O-gallate and rutin; (g) procyanidin B1 and quercetin; (h) procyanidin B2 and quercetin; (i) procyanidin B1 and isoquercitrin; (j) procyanidin B2 and isoquercitrin; (k) procyanidin B1 and rutin; and (1) procyanidin B2 and rutin.

In some embodiments, the effective amount is at least about 150 milligrams (mg). In some embodiments, the effective amount is about 150 mg or about 160 mg. In some embodiments, the effective amount ranges from about 150 mg to about 500 mg, including about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, and about 500 mg. In some embodiments, the composition comprises at least two of the compositions or compounds and the effective amount is at least about 150 milligrams (mg) in total of the two listed compositions or compounds, i.e., a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, (±)-epicatechin-3-O-gallate, (±)-epigallocatechin-3-O-gallate, procyanidin B1, procyanidin B2, quercetin, isoquercitrin, and rutin. Thus, in some embodiments, the composition comprises at least two of the listed compositions and the total weight of the at least two compositions is about 150 mg or at least about 160 mg. In some embodiments, the composition comprises at least two of the listed compositions and the effective amount of the combination of the two compositions ranges from about 150 mg to about 500 mg, including about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, and about 500 mg. In some embodiments, a combination comprising three or four compounds, or more, can also be provided, in some embodiments in a total amount of at least about 150 mg, optionally in an amount ranging from about 150 mg to about 500 mg, including about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, and about 500 mg. In some embodiments, the effective amount of any single composition or any combination of compositions is less than about 150 mg (e.g, about 140 mg, about 130 mg, about 120 mg, about 110 mg, about 100 mg, about 75 mg, about 50 mg, or less) or greater than about 500 mg (e.g., about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 900 mg, about 1000 mg or more). In some embodiments, the effective amount of the single composition or any combination of compositions is sufficient to provide about 2 mg/kg body weight subject/day to about 6.5 mg/kg body weight of subject/day, including about 2 mg/kg body weight subject/day, about 3 mg/kg body weight subject/day, about 4 mg/kg body weight subject/day, about 5 mg/kg body weight subject/day, about 6 mg/kg body weight subject/day about 6.5 mg/kg body weight subject/day.

In some embodiments, the presently disclosed subject matter provides a method of treating a viral infection in a subject in need of treatment thereof, wherein the method comprises administering to the subject an effective amount of a composition comprising a composition comprising one or more flavones, a composition comprising one or more flavanones, a composition comprising one or more flavonols, a composition comprising one or more dihydroflavonols, or any combination thereof. In some embodiments, the one or more flavones, the one or more flavanones, the one or more flavonols, and/or the one or more dihydroflavonols is provided as the active agent or agents against the virus causing the infection.

In some embodiments, the presently disclosed subject matter provides a composition for treating a viral infection in a subject in need thereof. In some embodiments, the composition comprises an effective amount of a composition comprising a composition comprising one or more flavones, a composition comprising one or more flavanones, a composition comprising one or more flavonols, a composition comprising one or more dihydroflavonols, or any combination thereof; and a carrier. In some embodiments, the one or more flavones, the one or more flavanones, the one or more flavonols, and/or the one or more dihydroflavonols is provided as the active agent or agents against the virus causing the infection.

In some embodiments, the presently disclosed subject matter provides methods and compositions for inhibiting main protease (M ^pro ) of SARS-CoV-2 and other coronavirus strains, wherein the method comprises contacting the main protease (M ^pro ) with an effective amount of a composition comprising a composition comprising one or more flavones, a composition comprising one or more flavanones, a composition comprising one or more flavonols, a composition comprising one or more dihydroflavonols, or any combination thereof. In some embodiments, the one or more flavones, the one or more flavanones, the one or more flavonols, and/or the one or more dihydroflavonols is provided as the active agent or agents against the SARS-CoV-2 virus.

In some embodiments, the presently disclosed subject matter provides methods and compositions for inhibiting hACE2 and SARS-CoV-2 S protein binding, optionally in a subject, optionally wherein a SARS-CoV-2 virus is causing COVID- 19 in the subject, and wherein the method comprises contacting the hACE2 and/or SARS-CoV-2 S protein binding with and/or administering to the subject an effective amount of a composition comprising a composition comprising one or more flavones, a composition comprising one or more flavanones, a composition comprising one or more flavonols, a composition comprising one or more dihydroflavonols, or any combination thereof. In some embodiments, the one or more flavones, the one or more flavanones, the one or more flavonols, and/or the one or more dihydroflavonols is provided as the active agent or agents against the SARS-CoV-2 virus.

In some embodiments, an effective amount of a composition comprising a composition comprising one or more flavones, a composition comprising one or more flavanones, a composition comprising one or more flavonols, a composition comprising one or more dihydroflavonols, or any combination thereof, is administered to the subject and/or is contacted with M ^pro of SARS-CoV-2 and/or is contacted with a hACE2 and/or SARS-CoV-2 S protein binding. In some embodiments, the hACE2 and SARS-CoV-2 S protein binding are present in a subject, such as wherein a SARS-CoV-2 virus is causing COVID-19 in the subject.

In some embodiments, the one or more flavones, the one or more flavanones, the one or more flavonols, and/or the one or more dihydroflavonols comprises a compound as set forth in Fig. 12 or one of Tables 1-3, above. In some embodiments, the one or more flavones is a single flavone molecule. In some embodiments, the one or more flavonols is a single flavonol molecule. In some embodiments, the one or flavanones is a single flavanone molecule. In some embodiments, the one or more dihydroflavonols is a single dihydroflavonol molecule. In some embodiments, the one or more flavones, the one or more flavanones, the one or more flavonols, and/or the one or more dihydroflavonols is selected from the group consisting of (+)-dihydroquercetin (DHQ), (+)-dihydrokaempferol (DHK), (+)-dihydroquercetin (DHM), kaempferol, quercetin, quercetin-3-O-gly coside (isoquercitrin), rutin, an analog of any of the foregoing compounds, and a derivative of any of the foregoing compounds or analog thereof.

In some embodiments, a combination of any two or more molecules listed above is used in a method or composition of the presently disclosed subject matter. In some embodiments, a combination of three or more molecules listed above is used in a method or composition of the presently disclosed subject matter.

The phrase “one or more flavones” includes derivatives and analogs of a flavone compound, including any flavone molecule specifically disclosed herein. The phrase “one or more flavanones” includes derivatives and analogs of a flavanone molecule, including any flavanone molecule specifically disclosed herein. The phrase “one or more flavonols” includes derivatives and analogs of a flavonol compound, including any flavonol compound specifically disclosed herein. The phrase “one or more dihydroflavonols” includes derivatives and analogs of a dihydroflavonol compound, including any dihydroflavonol compound specifically disclosed herein. These phrases specifically include all flavanone, and flavanonol flavone, and flavonol aglycones, their analogs, their glycosides, their gallates, acylates, and other derivatives, including those specifically disclosed herein. These phrases specifically encompass the compounds in Figure 12 and Tables 1-3.

In some embodiments, a composition in accordance with the presently disclosed subject matter comprises a molecule produced by metabolic engineering of plants, plant cells, plant organs, plant tissues, microbes, and/or enzymes.

In some embodiments, the viral infection is an infection of a virus of a virus family selected from coronaviridae, herpesviridae, retroviridae, papillomaviridae, caliciviridae, and picomaviridae. In some embodiments, the virus is selected from the group comprising a coronavirus (e.g., SARS, MERS, etc), HSV, HIV, HPV, a norovirus, a rhinovirus, Zika virus, an ebolavirus, and an influenza virus (e.g., influenza type B virus or influenza type C virus). In some embodiments, the virus is a coronavirus. In some embodiments, the virus is SARS- CoV-2. Thus, in some embodiments, the viral infection is a SARS-CoV-2 infection and the subject in need of treatment has COVID- 19.

In some embodiments, a subject is administered an effective amount of a second composition, wherein the second composition comprises a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, a composition comprising one or more flavan-3-ol compounds, a composition comprising one or more proanthocyanidin compounds, or any combination thereof. In some embodiments, the composition comprising a composition comprising one or more flavones, a composition comprising one or more flavonols, a composition comprising one or more dihydroflavonols, a composition comprising one or more flavanones, or any combination thereof and the second composition are formulated as a single composition. In some embodiments, the extract from green tea, the extract from grape berries and/or seeds, the extract from cacao and/or chocolate, the one or more flavan-3-ol compounds, and/or the one or more proanthocyanidin compounds is provided as the active agent or agents against a virus.

The phrase “one or more flavan-3-ol compounds” includes derivatives and analogs of a flavan-3-ol compound, including any flavan-3-ol molecule specifically disclosed herein. The phrase “one or more proanthocyanidin compounds” includes derivatives and analogs of a proanthocyanidin compound, including any proanthocyanidin compound specifically disclosed herein.

In some embodiments, the one or more flavan-3-ol compounds is a single flavan-3-ol- gallate molecule, such as but not limited to, (±)-afzelechin-3-O-gallate, (±)-epiafzelechin-3- O-gallate, (±)-catechin-3-O-gallate, (±)-epicatechin-3-O-gallate, (±)-gallocatechin-3-O- gallate, or (±)-epigallocatechin-3-O-gallate. In some embodiments, the one or more flavan-3- ol compounds is a single flavan-3-ol-3, 5-digallate molecule, such as (±)-afzelechin-3-O-3, 5- digallate, (±)-epiafzelechin-3-O-3, 5-digallate, (±)-catechin-3-O-3, 5-digallate, (±)- epicatechin-3-0-3, 5-digallate, (±)-gallocatechin-3-O-3, 5-digallate, or (±)-epigallocatechin- 3-0-3, 5-digallate.

In some embodiments, the one or more flavan-3-ol compounds is a single flavan-3-ol- sugar molecule, such as, (±)-afzelechin-3-O-gly coside, (±)-epiafzelechin-3-O- glycoside, (±)- catechin-3-0- glycoside, (±)-epicatechin-3-0gly coside, (±)-gallocatechin-3-O- glycoside, or (±)-epigallocatechin-3 -O- glycoside.

In some embodiments, the one or more flavan-3-ol compounds is a single flavan-3-ol- 3, 5-di-glycoside molecule, such as (±)-afzelechin-3-O-3, 5-di- glycoside, (±)-epiafzelechin- 3-0-3, 5- di- glycoside, (±)-catechin-3-O-3, 5- di-glycoside, (±)-epicatechin-3-O-3, 5- di- glycoside, (±)-gallocatechin-3-O-3, 5- di-glycoside, or (±)-epigallocatechin-3-O-3, 5- di- glycoside.

In some embodiments, the one or more proanthocyanidin compounds is a single dimeric procyanidin molecule, such as procyanidin B1, B2, B3, B4, B5, B6, B7, and B8, a dimeric propelargonidin, a dimeric prodelphinidins, or other miscellaneous proanthocyanidin. In some embodiments, a combination of any two molecules listed above is used. In some embodiments, a combination of three or more molecules listed above is used. In some embodiments, the composition comprises an extract from green tea, including any variety of green tea. In some embodiments, the composition comprises an extract from grape berries and/or seeds, including but not limited to any muscadine and/or venifera grape berries and/or seeds. In some embodiments, the composition comprises an extract from any cacao and/or any chocolate, including but not limited to dark chocolate.

In some embodiments, the presently disclosed subject matter provides technology for extraction of molecules disclosed herein from green tea, grape (e.g., muscadine) berry and seeds, cacao, and chocolate. In some embodiments, the presently disclosed subject matter provides for the production of molecules disclosed herein using metabolic engineering via the DFR-ANS-ANR pathway and UDP-flavan-3-ol glycosyltransferases, and UDP-flavan-3-ol acyltransferase in E. coli, yeast, any tobacco species, plants, plant organs, plant tissues, and plant cells.

By way of example and not limitation, the presently disclosed subject matter provides approaches to produce B-type and A-type PAs from engineered plants, muscadine gapes, tea, and cacao. An objective is to produce different B-type and A-type PAs for the anti-SARS- CoV-2 treatments disclosed herein. Except for procyanidin B1 and B2 and procyanidin Al and A2, most of the other PAs are not commercially available in the market. Full chemical synthesis of PAs is difficult. Therefore, PAs not commercially available can be obtained from plants by isolation. A red PAP1+ANR (P-A) tobacco plant has been engineered in which a full biosynthetic pathway of PAs has been created (Xie et al., 2006). These PA plants form a renewable bioreactor resource to produce PAs. Extraction of PAs and thin layer chromatography (TLC) analysis showed that PA plants produced flavan-3-ols, dimeric PAs, and high-degree oligomeric PAs in leaves. In addition, TLC shows more oligomeric PAs that can be isolated for anti-viral use. PA plants are grown in the greenhouse to isolate these compounds. Furthermore, a PA-producing cell type has been engineered from P+A leaves. The cultured cells express the PA biosynthetic pathway and produce flavan-3-ols and oligomeric PAs. A 4-liter scale cell suspension culture has been developed to produce flavan- 3-ols and Pas, as have extraction and TLC separation protocols (Xie et al., 2006). The purification protocol includes preparative TLC and HPLC separations. Firstly, TLC is used to fractionate compounds. Secondly, gradient elution program (comprising acetonitrile and 0.5% formic acid in HPLC-MS water) has been optimized to separate flavan-3-ols and Pas. This protocol is used to purify TLC fractions with a 25 x 2 cm (length by diameter) C18 silica column. In another representative, non-limiting example, compounds are isolated from muscadine grapes. In some embodiments, 100 pounds of berries are collected as a starting material. Recently, flavan-3-ols and PAs in two novel hybrids of muscadine grape, FLH 13- 11 and FLH 17-66, have been profiled. 4 Flavan-3-ols, 19 flavan-3-ol conjugates (including galloylated, glycosylated, and methylated ones), and 8 dimeric procyanidins including procyanidin Al and PB1, 2, 4, 5, 6, 7 and 8 were identified (Yuzuak et al., 2018).

In another representative, non-limiting example, flavan-3-ol-3, 5-digallates are produced for anti-SARS-CoV-2 tests and trials of COVID-19 therapy. Example digallates include (-)-epigallocatechin-3, 5-digallate (EGC-DG), (-)-epicatechin-3, 5-digallate (EC-DG), and gallocatechin-3, 5-digallate (GC-DG). Approaches to produce EGC-DG, EC-DG, GC- DG, and catechin-3, 5-digallate (CA-DG) include isolation from muscadine grape berry as described above. This is because FLH 13-11 and FLH 17-66 produce EC-DG. The same TLC and HPLC separation methods as described above are used.

In some embodiments, the presently disclosed subject matter provides methods and compositions for treating a viral infection in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, a composition comprising one or more flavan-3-ol compounds, a composition comprising one or more proanthocyanidin compounds, or any combination thereof.

In some embodiments, the presently disclosed subject matter provides methods and compositions for inhibiting main protease (M ^pro ) of SARS-CoV-2 and other coronavirus strains wherein the method comprises contacting the main protease (M ^pro ) with an effective amount of a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, a composition comprising one or more flavan-3-ol compounds, a composition comprising one or more proanthocyanidin compounds, or any combination thereof.

In some embodiments, the presently disclosed subject matter provides methods and compositions for inhibiting hACE2 and SARS-CoV-2 S protein binding, optionally in a subject, optionally wherein a SARS-CoV-2 virus is causing COVID- 19 in the subject, wherein the method comprises contacting the hACE2 and/or SARS-CoV-2 S protein binding with and/or administering to the subject an effective amount of a composition comprising an extract from green tea, a composition comprising an extract from grape berries and/or seeds, a composition comprising an extract from cacao and/or chocolate, a composition comprising one or more flavan-3-ol compounds, a composition comprising one or more proanthocyanidin compounds, or any combination thereof.

In some embodiments, an effective amount of a composition comprising one or more flavan-3-ol compounds, one or more proanthocyanidin compounds, or any combination thereof, is administered to the subject and/or is contacted with main protease (M ^pro ) of SARS- CoV-2 and/or is contacted with a (hACE2 and/or SARS-CoV-2 S protein binding. In some embodiments, the hACE2) and SARS-CoV-2 spike (S) protein binding are present in a subject, such as wherein a SARS-CoV-2 virus is causing COVID- 19 in the subject.

In some embodiments, the one or more flavan-3-ol compounds is a single flavan-3-ol- gallate molecule, such as but not limited to, (±)-afzelechin-3-O-gallate, (±)-epiafzelechin-3- O-gallate, (±)-catechin-3-O-gallate, (±)-epicatechin-3-O-gallate, (±)-gallocatechin-3-O- gallate, or (±)-epigallocatechin-3-O-gallate. In some embodiments, the one or more flavan-3- ol compounds is a single flavan-3-ol-3, 5-digallate molecule, such as (±)-afzelechin-3-O-3, 5- digallate, (±)-epiafzelechin-3-O-3, 5-digallate, (±)-catechin-3-O-3, 5-digallate, (±)- epicatechin-3-0-3, 5-digallate, (±)-gallocatechin-3-O-3, 5-digallate, or (±)-epigallocatechin- 3-0-3, 5-digallate.

In some embodiments, the one or more flavan-3-ol compounds is a single flavan-3-ol- sugar molecule, such as, (±)-afzelechin-3-O-gly coside, (±)-epiafzelechin-3-O- glycoside, (±)- catechin-3-0- glycoside, (±)-epicatechin-3-0gly coside, (±)-gallocatechin-3-O- glycoside, or (±)-epigallocatechin-3 -O- glycoside.

In some embodiments, the one or more flavan-3-ol compounds is a single flavan-3-ol- 3, 5-di-glycoside molecule, such as (±)-afzelechin-3-O-3, 5-di- glycoside, (±)-epiafzelechin- 3-0-3, 5- di- glycoside, (±)-catechin-3-O-3, 5- di-glycoside, (±)-epicatechin-3-O-3, 5- diglycoside, (±)-gallocatechin-3-O-3, 5- di-glycoside, or (±)-epigallocatechin-3-O-3, 5- diglycoside.

In some embodiments, the one or more proanthocyanidin compounds is a single dimeric procyanidin molecule, such as procyanidin B1, B2, B3, B4, B5, B6, B7, and B8, a dimeric propelargonidin, a dimeric prodelphinidins, or other miscellaneous proanthocyanidin.

In some embodiments, a combination of any two molecules listed above is used. In some embodiments, a combination of three or more molecules listed above is used. In some embodiments, the composition comprises an extract from green tea, including any variety of green tea. In some embodiments, the composition comprises an extract from grape berries and/or seeds, including but not limited to any muscadine and/or venifera grape berries and/or seeds. In some embodiments, the composition comprises an extract from any cacao and/or any chocolate, including but not limited to dark chocolate.

In some embodiments, the presently disclosed subject matter provides technology for extraction of molecules disclosed herein from green tea, grape (e.g., muscadine) berry and seeds, cacao, and chocolate. In some embodiments, the presently disclosed subject matter provides for the production of molecules disclosed herein using metabolic engineering via the DFR-ANS-ANR pathway and UDP-flavan-3-ol glycosyltransferases, and UDP-flavan-3-ol acyltransferase in E. coli, yeast, any tobacco species, and plant cells.

In some embodiments, the viral infection is an infection by a virus selected from the group comprising a coronavirus, HSV, HIV, HPV, a norovirus, a rhinovirus, Zika virus, an ebolavirus, and an influenza virus. The viral infection is a SARS-CoV-2 infection caused by any strain or variant thereof.

In accordance with one embodiment, a method for treating a subject in need of such treatment is provided. The method comprises administering a pharmaceutical composition to a subject in need thereof. Pharmaceutical and/or nutraceutical compositions useful for practicing the presently disclosed subject matter may be administered to deliver a dose or effective amount of between 1 ng/kg body weight subject/day and 100 mg/kg body weight subject/day. In some embodiments, the effective amount ranges from about 2 mg/kg body weight subject/day to about 6.5 mg/kg body weight of subject/day, including about 2 mg/kg body weight subject/day, about 3 mg/kg body weight subject/day, about 4 mg/kg body weight subject/day, about 5 mg/kg body weight subject/day, about 6 mg/kg body weight subject/day about 6.5 mg/kg body weight subject/day. In some embodiments, the presently disclosed subject matter provides a pharmaceutical and/or nutraceutical composition comprising an effective amount of at least about 150 mg, including about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, and about 500 mg. In some embodiments, the pharmaceutical and/or nutraceutical composition comprises an effective amount of less than about 150 mg (e.g, about 140 mg, about 130 mg, about 120 mg, about 110 mg, about 100 mg, about 75 mg, about 50 mg, or less) or greater than about 500 mg (e.g., about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 900 mg, about 1000 mg or more). The composition can be formulated in any suitable form for administration to the subject. In some embodiments, the composition is formulated for oral administration. In some embodiments, the composition is formulated as a capsule, a tablet, a pill, a sachet, or a drink. In some embodiments, the effective amount of the composition or combination of compositions is split between two or more (e.g., three, four, five or more) capsules, tablets, pills, sachets, drinks and/or any other suitable form. In some embodiments, the effective amount is provided in two or more different forms.

The presently disclosed subject matter encompasses the preparation and use of pharmaceutical and/or nutraceutical compositions comprising a compound useful for treatment of the diseases and disorders disclosed herein as an active ingredient. Such a pharmaceutical and/or nutraceutical composition may consist of the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical and/or nutraceutical composition may comprise the active ingredient and one or more carriers, including pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The active ingredient may be present in the pharmaceutical composition and/or nutraceutical in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

As used herein, the term “physiologically acceptable” ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical and/or nutraceutical composition, which is not deleterious to the subject to which the composition is to be administered.

For in vivo applications, the compositions of the presently disclosed subject matter may comprise a pharmaceutically acceptable salt. Suitable acids which are capable of forming such salts with the compounds of the presently disclosed subject matter include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid and the like; and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid and the like.

Pharmaceutically acceptable carriers include physiologically tolerable or acceptable diluents, excipients, solvents, or adjuvants. The compositions are in some embodiments sterile and nonpyrogenic. Examples of suitable carriers include, but are not limited to, water, normal saline, dextrose, mannitol, lactose or other sugars, lecithin, albumin, sodium glutamate, cysteine hydrochloride, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), vegetable oils (such as olive oil), injectable organic esters such as ethyl oleate, ethoxylated isosteraryl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methahydroxide, bentonite, kaolin, agar-agar and tragacanth, or mixtures of these substances, and the like.

The pharmaceutical and/or nutraceutical compositions may also contain minor amounts of nontoxic auxiliary pharmaceutical substances or excipients and/or additives, such as wetting agents, emulsifying agents, pH buffering agents, antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, and the like). Suitable additives include, but are not limited to, physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions (e.g., 0.01 to 10 mole percent) of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA or CaNaDTPA-bisamide), or, optionally, additions (e.g., 1 to 50 mole percent) of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). If desired, absorption enhancing or delaying agents (such as liposomes, aluminum monostearate, or gelatin) may be used. The compositions can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Pharmaceutical and/or nutraceutical compositions according to the presently disclosed subject matter can be prepared in a manner fully within the skill of the art.

Where the administration of the composition is by injection or direct application, the injection or direct application may be in a single dose or in multiple doses. Where the administration of the compound is by infusion, the infusion may be a single sustained dose over a prolonged period of time or multiple infusions.

The formulations of the pharmaceutical and/or nutraceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

A pharmaceutical and/or nutraceutical composition of the presently disclosed subject matter may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is a discrete amount of the pharmaceutical and/or nutraceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the carrier (e.g., pharmaceutically acceptable carrier), and any additional ingredients in a pharmaceutical and/or nutraceutical composition of the presently disclosed subject matter will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.

In addition to the active ingredient, a pharmaceutical and/or nutraceutical composition of the presently disclosed subject matter may further comprise one or more additional pharmaceutically active agents. Controlled- or sustained-release formulations of a pharmaceutical and/or nutraceutical composition of the presently disclosed subject matter may be made using conventional technology. As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical and/or nutraceutical compositions of the presently disclosed subject matter are known in the art and described, for example in Gennaro (1990) Remington’s Pharmaceutical Sciences. 18th ed., Mack Pub. Co., Easton, Pennsylvania, United States of America and/or Gennaro (ed.) (2003) Remington: The Science and Practice of Pharmacy. 20th edition Lippincott, Williams & Wilkins, Philadelphia, Pennsylvania, United States of America, each of which is incorporated herein by reference.

Typically, dosages of the compound of the presently disclosed subject matter which may be administered to an animal, in some embodiments a human, range in amount from 1 pg to about 100 g per kilogram of body weight of the animal. While the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of animal and type of disease state being treated, the age of the animal and the route of administration. In some embodiments, the dosage of the compound will vary from about 1 mg to about 10 g per kilogram of body weight of the animal. In another aspect, the dosage will vary from about 10 mg to about 1 g per kilogram of body weight of the animal.

The compositions may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the condition or disease being treated, the type and age of the animal, etc.

Suitable preparations include injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, suspension in, liquid prior to injection, may also be prepared. The preparation may also be emulsified, or the compositions encapsulated in liposomes. The active ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the preparation may also include minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants.

The formulations of the pharmaceutical and/or nutraceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

Compositions may be administered to, for example, a cell, a tissue, or a subject by any of several methods described herein and by others which are known to those of skill in the art.

A pharmaceutical or nutraceutical composition of the presently disclosed subject matter can be in the form of a solid or liquid. In some embodiments, the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form. In some embodiments, the carrier(s) can be liquid, with the compositions being, for example, oral syrup, injectable liquid or an aerosol, which is useful in, for example, for inhalatory administration.

The compositions described herein can be administered by either oral or non-oral pathways. When intended for oral administration, the pharmaceutical or nutraceutical composition is in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid. When administered orally, compositions can be administered in capsule, tablet, granule, spray, syrup, or other such form. Compositions also can be brewed, as with a tea, or formed by dissolving a powdered composition into a fluid, typically water, fruit or vegetable juice, or milk. Powdered compositions can also be sprinkled on or mixed in with a food item. Accordingly, the compositions can be mixed with water or an aqueous beverage including tea, milk, various soft drinks, coffee, smoothies, juice, other supplemental beverages, food supplements, candy, or food bars, and virtually any other food that can be supplemented with powder, liquid or gel.

In some embodiments, the presently disclosed compositions can be administered orally in a powder, gel, liquid, food bar, candy, sublingual tablet, or capsule form and can optionally be combined with a food or a drink as part of a treatment regimen. When provided to nonhumans, the compositions of the presently disclosed subject matter can be administered separately or can be combined with ordinary feed or liquid nourishment. The presently disclosed compositions, including the extracts described herein, can be provided in oral dosage form, or in combination with any of the usual pharmaceutical or nutritional media employed in the art for oral liquid preparations, e.g., suspensions, elixirs, and solutions. Generally, media containing water, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can also be used for flavor, texture, or shelf-life enhancement. Carriers such as starch, glycerol, alcohol, sugars, diluents, granulating agents, flow agents, lubricants, binders, disintegrating agents, and the like can be used to prepare oral solids (e.g., a solid cake, powders, capsules, pills, and sublingual tablets). Controlled release forms can also be used. Because of their ease in administration, tablets, pills, and capsules represent advantageous oral dosage unit forms, in which case solid pharmaceutical carriers are employed. If desired, tablets can be sugar- coated or enteric-coated by standard techniques.

As a solid composition for oral administration, the pharmaceutical or nutraceutical composition can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer, bar, or like form. Such a solid composition will typically contain one or more inert diluents or edible carriers which can be, for example, in particulate form. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethyl cellulose, cyclodextrin, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, a sodium starch glycolate, com starch and the like; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent.

When the pharmaceutical or nutraceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.

In some embodiments, the pharmaceutical or nutraceutical composition can be in the form of a liquid, for example, an elixir, syrup, gel, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, a useful composition contains, in addition to the present compounds or compositions, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer can be included. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can be included.

The liquid pharmaceutical or nutraceutical compositions of this disclosure, whether they be solutions, suspensions or other like form, can include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, such as physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as carbonate, citrates, acetate, lactate, gluconate, or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a generally useful adjuvant. An injectable pharmaceutical or nutraceutical composition is sterile.

The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the presently disclosed subject matter will vary, depending upon the identity, sex, age, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered.

Other components such as preservatives, antioxidants, surfactants, absorption enhancers, viscosity enhancers or film forming polymers, bulking agents, diluents, coloring agents, flavoring agents, pH modifiers, sweeteners or taste-masking agents may also be incorporated into the composition. Suitable coloring agents include red, black, and yellow iron oxides and FD&C dyes such as FD&C B1ue No. 2, FD&C Red No. 40, and the like. Suitable flavoring agents include mint, raspberry, licorice, orange, lemon, grapefruit, caramel, vanilla, cherry grape flavors, combinations thereof, and the like. Suitable pH modifiers include citric acid, tartaric acid, phosphoric acid, hydrochloric acid, maleic acid, sodium hydroxide, and the like. Suitable sweeteners include aspartame, acesulfame K, thaumatic, and the like. Suitable taste-masking agents include sodium bicarbonate, ion-exchange resins, cyclodextrin inclusion compounds, adsorbates, and the like.

Although the descriptions of pharmaceutical and/or nutraceutical compositions provided herein are principally directed to pharmaceutical and/or nutraceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical and/or nutraceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical and/or nutraceutical compositions of the presently disclosed subject matter is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs, and birds including commercially relevant birds such as chickens, ducks, geese, and turkeys.

As used herein, an “instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the methods of the presently disclosed subject matter in a kit for effecting the treatments disclosed herein. Optionally, or alternately, the instructional material may describe one or more methods of using the compositions for therapeutic purposes or of alleviation the diseases or disorders in a cell or a tissue of a mammal. The instructional material of the kit of the presently disclosed subject matter may, for example, be affixed to a container which contains one or more reagents for carrying out the presently disclosed subject matter or be shipped together with a container which contains the one or reagents. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the reagents be used cooperatively by the recipient.

In accordance with the presently disclosed subject matter, as described above or as discussed in the Examples below, there can be employed conventional chemical, cellular, histochemical, biochemical, molecular biology, microbiology, recombinant DNA, and clinical techniques which are known to those of skill in the art. Such techniques are explained fully in the literature. See for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Publications, Cold Spring Harbor, New York, United States of America; Glover (1985) DNA Cloning: A Practical Approach. Oxford Press, Oxford; Gait (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford, England; Harlow & Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York; Roe et al. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley, New York, New York, United States of America; and Ausubel et al. (1995) Current Protocols in Molecular Biology, Greene Publishing.

The presently disclosed subject matter may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The presently disclosed subject matter encompasses all combinations of the different aspects of the presently disclosed subject matter noted herein. It is understood that any and all embodiments of the presently disclosed subject matter may be taken in conjunction with any other embodiment or embodiments to describe additional representative embodiments. It is also to be understood that each individual element of the disclosed embodiments is intended to be taken individually as its own independent representative embodiment. Lurthermore, any element of an embodiment is meant to be combined with any and all other elements from any embodiment to describe an additional embodiment. EXAMPLES

The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter.

To seek effective treatment and prevention of COVID- 19, virtual screening of flavan- 3-ol and PA compounds was performed. Based on positive virtual screening results, the M ^pro of SARS-CoV-2 was used as a target to perform in vitro inhibition experiments. Two compounds were initially identified to have strong anti- M ^pro activity. Crude extracts from early spring green tea, cacao, and muscadine grape, which include these two compounds, showed effectively inhibitory effects on the M ^pro activity. These results suggest that crude extracts of these three plant products and dark chocolate can be used to treat or prevent COVID-19 and/or SARS-CoV-2 infection.

EXAMPLE 1

Candidates of flavan-3-ols and PAs for virtual analysis

Main flavan-3-ol aglycones include (±)-afzelechin, (±)-epiafzelechin, (±)-catechin (CA), (±)-epicatechin (EPC), (±)-gallocatechin, and (±)-epigallocatechin (EGC). Galloylated flavan-3-ol conjugates include (±)-epicatechin-3-O-gallate (EPCG), (±)-catechin-3-O-gallate (CAG), (±)-gallocatechin 3-O-gallate (GCG), and (±)-epigallocatechin-3 -gallate (EGCG). Dimeric proanthocyanidins include procyanidin Al, A2, B1, and B2. Their structures are shown in Figures 2 and 3. In addition, three known anti-viral compounds, ebselen, cinanserin, and lopinavir, were used as positive metabolite control.

EXAMPLE 2

Plant materials

Three types of plant products rich in flavan-3-ols and dimeric PAs were used for extraction. “Xin-Yang-Mao-Jian” is one of famous green tea (Camellia sinensis) products in China. This product is composed of newly leaves (0.8-1.2 cm in length) of early spring sprouts (harvested around April 5 every year). This green tea product was produced in 2019. Cacao (Theobroma cacao) seed powder used were obtained from Ecuador in 2019. Ripen muscadine grape berries of FLH 13-11 and FLH 17-66 were collected in 2011 and 2012, ground into powder in liquid nitrogen, freeze dried, and stored in -20°C freezer (Yuzuak et al. 2018). EXAMPLE 3 M ^pro docking analysis

M ^pro was used to perform docking simulation via two publicly available software packages, the Dock Prep tool of UCSF-Chimera (available online at www.cgl.ucsf.edu/chimera/docs) and AutoDock Vina (available at vina.scripps.edu). In addition, three reported potential anti-COVID-19 candidates, ebselen (Jin et al. 2020a), cinanserin (Jin et al. 2020b; Chen et al. 2005), and lopinavir (Cao et al. 2020), were used as controls. Four steps were used to complete docking. First, a SARS-CoV-2 M ^pro (PDB ID: 6LU7) structure was obtained from the Protein Data Bank (available at rcsb.org). The structure included M ^pro and an inhibitor N3 (Jin et al. 2020a). See Figure 4B. Second, the Dock Prep tool of UCSF-Chimera was used to perform receptor (M ^pro ) preparation. During the receptor preparation, the inhibitor N3 was removed. In addition, hydrogens were added and receptor charge was optimized, which allowed determining the histidine protonation state. The resulting M ¹ ”” structure file was saved in the mol2 format. Third, the 3D structures of flavan- 3-ols and other interesting compounds used in this study were obtained from PubChem (pubchem.ncbi.nlm.nih.gov) and used as ligands. All ligand structures were then uploaded to the Minimize structure tool of UCSF-Chimera and minimized, during which charges and hydrogens were added to each ligand. The resulting ligand files were also saved in the mol2 format. Fourth, both M ^pro and ligand files were uploaded to AutoDock vina for docking. During modelling, the M ^pro protein was framed in one box for receptor-ligand docking. The entire box size was x=50, y=55, and z=50 and the origin center of the box was at x=-27, y=13, and z=58.

Furthermore, a docking analysis was completed with human angiotensin-converting enzyme 2 (hACE2), SARS-CoV-2 spike (S) protein and four compounds. The resulting data showed that four compounds EGC, EGCG, EPC-DG, and EGC-DG can bind to the interacting space of hACE2 and S proteins. These data show that flavan-3-ols and PAs are inhibitor candidates to block the binding of hACE2 to the S protein.

EXAMPLE 4

Extraction of flavan-3-ols and PAs from tea, cacao, and muscadine berry powders

One gram of powder was suspended in 10 ml acetone: deionized water (70:30) contained in a 50 ml falcon tube. The tube was strongly vortexed for 5 min and then sonicated in a water bath for 10 min at the room temperature, followed by centrifugation at 4000 rpm for 20 min. The resulting supernatant was pipetted to a new 50 ml tube. The remained pellet was extracted again following the same steps. Two extractions were pooled together to obtain 20 ml in volume, which was reduced to 5 ml by removing acetone with a nitrogen gas flow at the room temperature. The tube containing the remained water phase was added 1.0 ml chloroform and then strongly vortexed for 2 min, followed by centrifugation at 4000 rpm for 5 min. The resulting lower chloroform phase containing non-polar compounds was removed. This step was repeated once. The tube that contained the remained water phase including flavan-3-ols and PAs was added 5 ml ethyl acetate, strongly vortexed for 3 min, and then centrifuged at 4000 rpm for 5 min. The resulting upper ethyl acetate phase containing flavan- 3-ols and PAs was transferred to a new 50 ml tube. This ethyl acetate extraction step was repeated two times. The three times of ethyl acetate extractions were pooled together and then dried completely with nitrogen gas flow at the room temperature. The remained residue was dissolved in dimethyl sulfoxide (DMSO) to obtain 50 mg/ml extracts and stored in a -20°C freezer until use.

EXAMPLE 5

In vitro inhibition assay ofM ^pro activity

(+)-CA, (-)-EC, (-)-GC, (-)-EGC,(-)-ECG, (-)-EGCG, (+)-CAG, (-)-GCG, procyanidin A2 (PA2), and procyanidin B2 (PB2) were purchased from Sigma-Aldrich (St. Louis, Missouri, United States of America) and dissolved in DMSO to prepare 1.0 mM stock solution. A 3 CL Protease (M ^pro ) (SARS-Cov-2) Assay Kit (BPS Bioscience, San Diego, California, United States of America), was used to test the inhibitory activity of these ten compounds. The steps of in vitro assay followed the manufacturer’s protocol. In brief, each reaction was completed in a 25 pl volume in 96-well plates. Each reaction solution contains 150 ng recombinant M ^pro (6 ng/ pl), 1 mM DDT, 50 μM fluorogenic substrate, and (+)-CA, (- )-EC, (-)-GC, (-)-EGC,(-)-ECG, (-)-EGCG, (+)-CAG, (-)-GCG, PA2, and PB2 (0, 0.1, 0.5, 1, 5, 10, 50, 100, 150, and 200 μM) or crude plant extracts (0, 1, 10, 100, 1000 pg/ml) in pH 8.0 50 mM Tris-HCl and 5μM EDTA buffer. GC376 (50 μM) was used as positive control and Tris-HCl-EDTA buffer was used as negative control. The reaction mixtures were incubated for 4 hours at the room temperature. The fluorescence intensity of each reaction was measured and recorded on a microtiter plate-reading fluorimeter (sold under the tradename SYNERGY™ H4 Plate Reader, BioTek, Winooski, Vermont, United States of America) for detect fluorescent and luminescent signals. The excitation wavelength was 360 nm and the detection emission wavelength was 460 nm. Each concentration of all compounds and extracts was tested five times. A mean value was calculated using five individual replicates. One-way analysis of variance (ANOVA) was performed to evaluate the statistical significance. The P- value less than 0.05 means significant differences.

Examples 1-5 - Results and Discussion

Theoretical compound screening for inhibitors of the SARS-CoV-2 M ^pro is an effective approach to identify potential candidates that can be used for trials to test their inhibitory activity against SARS-CoV-2 (Zhang et al. 2020a; Jin et al. 2020c; Xue et al. 2008; Pillaiyar et al. 2016; Dai et al. 2020a). To understand whether flavan-3-ol and oligomeric PA molecules (see Figures 2 and 3) could inhibit the M ^pro activity, docking analysis was performed via two publicly available software packages, Dock Prep tool of UCSF-Chimera and AutoDock vina). The resulting docking data showed that all flavan-3-ol aglycones, flavan-3-ol gallates, and dimeric procyanidins tested (B1, B2, A2 and A2) (see Figures 2 and 3) could bind to the same location of M ^pro bound by N3 inhibitor. See Figures 4A-4F and Figure 5. In addition, two antiviral control compounds, ebselen and cinanserin, bound to the same binding pocket. See Figure 4G. In contrast, the positive antiviral medicine, lopinavir, bound to a different location. See Figure 4H. The resulting affinity scores were -7.0 to -7.7 for (+)-afzelechin (AF), (-)- epiafzelechin (EAF), (+)-catechin (CA), (-)-epicatechin (EPC), (+)-gallocatechin (GC), and (- )-epigallocatechin (EGC), -8.3 for (+)-catechin-3-O-gallate (CAG), -8.7 for (-)-gallocatechin- 3-O-gallate (GCG) and (-)-epigallocatechin-3-O-gallate (EGCG) , and -9.2 for procyanidin B1, B2, Al and A2. See Table 7, below. All these score values of tested flavan-3-ols and PAs were lower than those of ebselen and cinanserin, -6.6 and -5.4. The affinity scores of dimeric procyanidins and flavan-3-ol-gallates were lower than -8.0 for lopinavir. These data suggested that flavan-3-ols and dimeric PAs could inhibit the M ^pro activity.

The substrate binding pocket of M ^pro has four subsites, S1’, S1, S2, and S4 (Dai et al. 2020a). Substrate-binding analysis was completed for 14 compounds and ebselen. The results showed different binding features among flavan-3-ol aglycones, flavan-3-ol-gallates, and dimeric PAs. See Figures 6A-6F. The A-ring and B ring of EGC bound to S2/S4 and S1, respectively. See Figure 6B. AF, EAF, CA, EPC, and GC also bound to these subsites. The A-ring, B-ring, and gallate ester group of EGCG bound to S2, S1’ and S1, respectively. See Figure 6C. CAG, EPCG, and GCG also bound to these subsites. EGC-DG bound to four subsites featured by the A-ring to S2, the B-ring to S1’, 3-gallate to S1, and 5-gallate to S4. See Figure 6D. In procyanidin Al and A2 binding, the A-ring and B-ring of the starter unit and the B-ring of the upper unit bound to S1, S4, and S1’, respectively. See Figure 6E. In procyanidin B1 and B2 binding, the B-ring of the starter unit and the A-ring and B-ring of upper unit bound to S1’, S1 and S2, respectively. See Figure 6F.

This docking analysis also predicted that the binding of these compounds to the substrate pocket was via the formation of hydrogen bonds. At the best affinity score of each compound (see Table 7), the modeling could predict the most potential hydrogen bonds. The resulting data showed different features of the number of hydrogen bonds and potential linkage between ligands and amino acids of M ^pro . See Figures 7A-7E. AF and M ^pro were predicted to form three hydrogen bonds via 7-O-Leul41, Oi and glutamic acid 166 (Glul66), and 3-0- Glul66. See Figure 7A. EAF and AF ^ro were predicted to form three hydrogen bonds via 7- O-Leul41, O1-Glu166, and 4’-O-Glnl89. See Figure 7A. CA and M ^pro were predicted to form three hydrogen bonds via Oi-Glul66, 3-O-Glul66, and 4’-O-Leul41. See Figure 7B. EPC and M ^pro were predicted to form three hydrogen bonds via Oi-Glul66, 7-O-Thrl90, and 4’-0-Glnl 89. See Figure 7B. GC and AF ^ro were predicted to form one hydrogen bond via Oi- Glul66. See Figure 7C. EGC and AF ^ro were predicted to form one hydrogen bond via Oi- Glul66. See Figure 7C. GCG and AF ^ro were predicted form one hydrogen bond via Oi- Glul66. See Figure 7D. EGCG and AF ^ro were predicted to form four hydrogen bonds via C7- O-Thrl90, Oi-Glul66, C ₅ -O-Phe140, and gallate-O-Glyl43. See Figure 7D. Procyanidin Al and AF ^ro were predicted to form one hydrogen bond via C ₅ -O-Glyl43. See Figure 7E. Procyanidin B2 and AF ^ro were predicted to form three hydrogen bounds via C ₅ -O-Glyl43 and C ₅ --O-Cysl45 on the B ring of the upper unit and C ₅ -O-Glul66 on the C-ring of the starter unit. See Figure 7E. Furthermore, the hydrogen-binding distances were different between ligands and AF ^ro .

To further test the inhibitory effects of these compounds on the AF ^ro activity, an in vitro inhibition assay was performed using CA, EPC, GC, EGC, CAG, ECG, GCG, EGCG, procyanidin A2, and procyanidin B2. The resulting data showed that CAG, ECG, GCG, EGCG, and procyanidin B2 inhibited the M ^pro activity. See Figures 8A-8D and 8G. The values of the half maximal inhibitory concentration (IC50) for CAG, ECG, GCG, EGCG and B2 were approximately 2.98, 5.21, 6.38, 7.5 and 75.3 μM, respectively. See Figures 8A-8F and 8G. In contrast, the results did not show that EGC, CA, EPC, GC, and procyanidin A2 in a range of concentrations from 0-500 μM tested could inhibit the AF ^ro activity. See Figures 8E-8F and Figures 9A-9D. It was interesting that when their concentrations were increased, such as 100 μM, the inhibition of procyanidin B2 was more efficient than that of EGCG. See Figure 8F. These data indicated that CAG, ECG, GCG, EGCG and procyanidin B2 inhibited the AF ^ro activity likely via their binding configurations. See Figures 6C and 6F.

Green tea, dark chocolate, cacao, and grape berry (particularly seeds and skin) are rich in flavan-3-ols and dimeric PAs. To show whether they can enhance the improvement of COVID-19 or prevent SARS-CoV-2 infection, these products were extracted and the extracts tested for inhibition against the AF ^ro activity. The results showed that the AF ^ro activity was inhibited by extracts of these products. See Figure 10A-10F. The green tea extracts showed the most inhibitory activity with a low IC50 2.84±0.25 pg/ml. When at 10 pg/ml tested, the extract of green tea completely inhibited the M ^pro activity. See Figure 10A. The strong inhibition likely resulted from the high content of EGCG and relatively high contents of ECG, CAG, and dimeric procyanidin B1, B2, B3, and B4 produced in green tea (Zhao et al. 2017; Wang et al. 2020c). The extracts of two muscadine grapes FLH 13-11 and FLH17-66 strongly inhibited the M ^pro activity with IC50 29.54±0.41 pg/ml and IC50 29.93±0.83 pg/ml. At 100 pg/ml, extracts of two muscadine grapes completely inhibited the M ^pro activity. See Figures 10B and 10C. The inhibition resulted from the production of EGCG, ECG, and procyanidin B1-B3 and B5-B8 (Yuzuak et al. 2018). Although the inhibition of cacao and dark chocolate extracts was not effective as both green tea and muscadine grape extracts, two at higher than 10 pg/ml showed promising inhibitory effects on the M ^p '° activity withIC ₅₀ 153.3 ±47.3 pg/ml and IC50256.39 ±66.3 pg/ml, respectively. See Figures 10D and 10E. The inhibition of cacao and dark chocolate extracts also resulted from the production of ECG, EGCG, and procyanidin B2 in these products (Schewe et al. 2001; Takahashi et al. 1999). Inhibitory activity of all extracts was compared at 100 pg/ml. The results showed that the extracts of green tea and muscadine grapes completely inhibited the M ^pro activity and the extracts of cacao and dark chocolate reduced the M ^pro activity by 40-50%. See Figures 10F.

Furthermore, a docking analysis was completed with human human angiotensinconverting enzyme 2 (hACE2), SARS-CoV-2 spike (S) protein and four compounds. The resulting data showed that four compounds EGC, EGCG, EPC-DG, and EGC-DG can bind to the interacting space of hACE2 and S proteins. See Figures 11A-1 ID. These data show that flavan-3-ols and PAs are inhibitor candidates to block the binding of hACE2 to the S protein.

In summary, given that there is not an effective medicine for the treatment of COVID- 19 and prevention against the SARS-CoV-2 infection and transmission, an effective strategy would use pharmaceuticals presented in food and beverage products to fight against the viral infections and COVID- 19. Based on the presently disclosed findings, extracts from green tea, grape, and cacao can be used to interfere with the devastation of SARS-CoV-2. For example, one approach is to eat dark chocolate and commercial products of these plants two-three times a day. Table 7. The affinity scores of AF, EAF, CA, EPC, GC, EGC, GCG, EGCG, PA2 and PB2 and three putative anti-COVID-19 molecules binding to the main protease.

Materials and methods for EXAMPLES 6-8

Dihydroflavonols and flavonols

Flavonols used in this study included kaempferol, quercetin, myricetin, quercetin-3- O-glycoside, and rutin. Dihydroflavonols used were (+)-taxifolin (dihydroquercetin, DHQ), (+)-dihydrokaempferol (DHK), and (+)-dihydromyricetin (DHM). The ebselen was used as a positive control. Two flavan-3-ols, (-)-epicatechin and (+)-catechin, were used as negative compound controls.

M ^pro docking simulation

The docking simulation of flavan-3-ols is described above. The same steps were used for docking simulation of flavones, flavonones, flavonols and dihydroflavonols in this experiment. In brief, three main steps were used, protein preparation, ligand preparation, and protein-ligand docking. The first was protein preparation. The SARS-Cov-2 M ^pro was used as a receptor to test ligands. Its ID is PDB ID: 6LU7 at Protein Data bank (www.rcsb.org), from which its 3D structure was downloaded to a desktop computer and then was prepared as a receptor of ligand via the Dock Prep tool of UCSF-Chimera (www.cgl.ucsf.edu/chimera). Because M ^pro contains the inhibitor peptide N3, we removed N3 prior to docking simulation. Hydrogens and charges were added and optimized to allow determining the histidine protonation state. The second step was ligand preparation. The 3D structures of compounds were obtained from PubChem (pubchem.ncbi.nlm.nih.gov) and then used as ligands. All structures were minimized by using the minimize structure tool of UCSF-Chimera. Hydrogens and charges were added to the ligands, which were then saved as mol2 format for the proteinligand docking simulation. The third step was protein-ligand docking. The modeling of protein-ligand docking was performed via the publicly available AutoDock vina (vina.scripps.edu) software. The protein and ligand files were loaded to the AutoDock vina through the UCSF-Chimera surface binding analysis tools. A working box was created to contain the whole receptor. The box center was set at x =-27, y = 13, and z = 58. The box size was set as x = 50, y =55, and z = 50, which framed the entire receptor to allow free position changes and ligand binding to the receptor at any potential positions.

M ^pro activity inhibition assay

(+)-DHQ, (+)-DHK, (+)-DHM, quercetin, kaempferol, myricetin, quercetin-3-O- glycoside, rutin, (-)-epicatechin, and (+)-catechin were purchased from Sigma-Aldrich (St. Louis, Missouri, United States of America) and dissolved in DMSO to prepare 1.0 M solution. An (SARS-Cov-2) Assay Kit (BPS Bioscience, San Diego, California, United States of America) was used to test the inhibitory activity of these compounds. Structures are shown in Figure 12. The steps of in vitro assay followed the manufacturer’s protocol as performed in Example 5, above. In brief, each reaction was carried out in a 25 pl volume in 384-well plates. Each reaction solution contains 150 ng recombinant M ^pro (6 ng/pl), 1 mM DDT, 50 μM fluorogenic substrate, and one compound (0, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 150, and 200 μM) in pH 8.0 mM Tris-HCl and 5 μM EDTA buffer. GC376 (50 μM) was used as positive control, while (-)-epicatechin and (+)-catechin were used as two negative controls. The reaction mixtures were incubated for 2 hrs at room temperature. The fluorescence intensity of each reaction was measured and recorded on a microtiter plate-reading fluorimeter (sold under the tradename SYNERGY™ H4 Plate Reader, BioTek, Winooski, Vermont, United States of America) to detect fluorescent and luminescent signals. The excitation wavelength was 360 nm and the detection emission wavelength was 460 nm. Each concentration of each compound was tested five times. A mean value was calculated using five individual replicates. One-way analysis of variance (ANOVA) was performed to evaluate the statistical significance. The P- value less than 0.05 means significant differences. EXAMPLE 6

Ligand-receptor docking of flavonols and dihydroflavonols to M ^pro

Docking simulation was completed with the UCSF-Chimera and AutoDock Vina software to evaluate the binding abilities of flavonols and dihydroflavonols to the SARS-Cov- 2 M ^pro . The M ^pro structure has a substrate-binding pocket which is shown in a red rectangle in Fig. 13. When the 3D structure of the protein was downloaded from the public database, the peptide inhibitor N3 was shown to bind to this pocket. During protein preparation, N3 was removed for docking. The simulation results showed that (+)-DHQ, (+)-DHK, (+)-DHM, quercetin, kaempferol, myricetin, quercetin-3-O-gly coside, rutin, (-)-epicatechin, (+) catechin, and ebselen bound to the binding pocket. See Figure 13.

The resulting affinity scores for (+)-DHQ, (+)-DHK, (+)-DHM, quercetin, kaempferol, myricetin, quercetin-3-O-gly coside, and rutin ranged from -7.4 to -8.8, better than the score of ebselen (-6.6). See Table 8, below. The flavone and flavonone skeletons have a close score as ebselen. See Table 8. The scores among the aglycones of (-)-epicatechin, (+)-catechin, three dihydroflavonols, and three flavonol aglycones were close, either -7.4 or -7.5. These data suggested that flavones, flavonones, dihydroflavonols, flavonols, and glycolated flavonols can inhibit the M ^pro activity.

EXAMPLE 7

Docking features at the binding pocket of M ^pro

The M ^pro substrate-binding pocket includes four subsites, S1’, S1, S2, and S4. The Cys 145 is a key residue located at the space among subsites S1, S1’, and S2 (Jin et al., 2020c, Dai et al., 2020). See also Figure 14A. Several studies have reported that the thiol of the Cys 145 is involved in M ^pro function and a binding to this residue can inhibit the M ^pro activity (Ramajayam et al., 2011, Dai et al., 2020, Chen et al. 2005). When ebselen was used as a positive compound for simulation again, it bound to this residue featured by three rings facing to the S1 and S1’ subsites. See Figure 14B. The docking simulation results showed that three dihydroflavonols, three flavonols aglycones, and two glycosylated flavonols bound to 2-4 subsites via the Cysl45 residue. In three dihydroflavonols tested, (+)-DHK and (+)-DHQ showed a difference in their occupation in the binding site. The A and B rings of (+)-DHK and (+)-DHQ dwelled in the S1’ and S2 subsites and their heterocycle C ring resided in the space between the S1 and S2 subsites. See Figures 14C and 14D. The A and B rings of DHM occupied the S1 and S4 subsites and the heterocycle C ring resided in the space between the S1 and S2 subsites. See Figures 14E. In three flavonol aglycones tested, the occupation of kaempferol was different from those of quercetin and myricetin. The A-ring, B-ring, and heterocycle C-ring of kaempferol resided in the S1’, S2, and the space between S1 and S2, respectively. See Figure 14F. The A-ring, B-ring, and the heterocycle C-ring of quercetin and myricetin dwelled in the S1, S4, and the space between S1 and S2. See Figures 14G and 14H. In comparisons, the residing positions of isoquercitrin and rutin were more complicated. The A-ring, B-ring, heterocycle C-ring, and 3-O-glucose of isoquercitrin occupied the S2, S1’, the space between S1 and S2, and S1 sites. See Figure 141. The A-ring, B-ring, heterocycle C- ring, 6-β-glucopyranose, and 1-L-α-rhamnopyranose of rutin occupied S4, S1’, the space between S1/S2, S1, and S4. See Figure 14J. These occupations in the binding sites suggested that these compounds might have an inhibitive activity against M ^pro .

EXAMPLE 8

In vitro inhibitory effects of four flavanone and flavones on the M ^pro activity

(+)-DHQ, (+)-DHM, quercetin, kaempferol, myricetin, isoquercetin (quercetin-3-O- gly coside), and rutin were used to test their inhibitory effects on the M ^pro activity. In addition, (-)-epicatechin and (+)-catechin were used as negative controls. The resulting data showed that (+)-DHQ, (+)-DHK, (+)-DHM, quercetin, kaempferol, myricetin, isoquercetin (quercetin-3- O-glycoside), and rutin inhibited the M ^pro activity. The half maximum inhibitory concentrations (IC50) were 0.125-12.94 μM. See Figures 15A-15G. Among the seven compounds tested, rutin had the lowest IC50 value with the most effective activity to inhibit the M ^pro (see Fig. 15G), while (+)-DHQ had the highest IC50 value with the lowest inhibitive activity. See Figure 15D. One hundred μM was used to further compare the inhibitive effects of these compounds on the M ^pro activity in a given time. The resulting data showed the most effectiveness of rutin. See Figure 15H. In addition, as reported previously, (+)-catechin and (-)-epicatechin did not show an inhibitory effect on the M ^pro activity in the range of concentrations from 0-200 μM. For example, these two compounds did not show any inhibitive activity at 100 μM. See Figure 15H.

Discussion of EXAMPLES 5-8

The development of medicine can complement the use of vaccines to control COVID- 19. The SARS-CoV-2 M ^pro is one of the targets to screen, repurpose, or develop drugs to treat or prevent SARS-CoV-2 (Ramajayam et al., 2011, Ren et al., 2013, Dai et al., 2020). One strategy to inhibit the M ^pro activity is to deliver a compound to the CYS145 at the space crossing the region of S1’ and S1 subsites (Dai et al., 2020). Ebselen is a small molecule candidate that has been found to inhibit the M ^pro activity with an IC50 0.46 μM (Jin et al., 2020c). Its molecular structure with three rings was revealed to be an effective vessel to deliver its carbonyl group to Cys 145. See Figure 14B. As described above, epicatechin gallate, epigallocatechin gallate, gallocatechin gallate, catechin gallate, and procyanidin B2 can effectively inhibit the activity of M ^p '° via the formation of hydrogen bonds with different amino acids in the binding pocket. These findings indicate that a peptide bond could be another potential alternative strategy to screen more flavonoids to intervene in COVID- 19. In this study, a docking simulation of flavonols and dihydroflavonols was performed. These compounds (see Figure 12) have C-4 keto and 1-0 structures in the heterocycle C-ring. Like flavan-3-ol gallates, the structures of these two groups can reside in the space S1 and S2 subsites. Ligand-docking simulation showed that these compounds could bind to the substratebinding pocket of M ^pro and occupied their heterocyclic C ring in the crossing region between S1 and S2. Furthermore, the docking results predicted the A-ring and B-ring of two, three, two, and one compounds could bind to S1’ and S2, S1 and S4, S2 and S1’, and S4 and S1’, respectively. See Figures 14C-14J. The docking results further showed that a glycosylation of quercetin increased the dwelling capacity in the binding site. Rutin was predicted to occupy all four subsites. See Figure 14J. The increase of binding subsites was also reflected by the affinity scores of M ^pro -ligands. Rutin and isoquercitrin (quercetin-3-O-glycoside) had the lowest and second lowest scores. See Table 8. These data indicate that not only might these compounds have an inhibitive activity but also a lower affinity score can have a better inhibition against the M ^pro activity. Further in vitro assays substantiated the prediction of docking simulation. Seven available compounds inhibited the activity of M ^pro with IC50 values from 0.125 to 12.9 μM. These data suggest that these compounds have a therapeutic application.

Quercetin, isoquercitrin, and rutin are three common supplements given that their nutritional values benefit human health. The presently disclosed subject matter suggests that quercetin, isoquercitrin, and rutin can be helpful to intervene in COVID- 19. These compounds are plant natural flavonoids and their bio-availability, metabolism, and toxicity have been studied extensively (Hostetler et al., 2017). In general, these compounds are safe nutrients sold as supplements or in food products such as onion and common dinner table fruits (Burak et al., 2017; Egert et al., 2012; Careri et al., 2003; Meng et al., 2004; Erlund et al., 2002; Snyder et al., 2016. Further, quercetin can be absorbed into the human body from the intestines. A large number of human health studies have reported the presence of quercetin and its derivatives in the blood plasma and their nutritional benefits after consumption (Day and Williamson, 2001; Shi and Williamson, 2016; Mohammadi-Sartang et al., 2017; Huang et al., 2020). [45-48], For example, the quercetin concentration in plasma was reported to reach 5.0±1.0 μM after the intake of 150 mg in one hour (Olthof et al., 2000; de Whalley et al., 1990). In addition, these compounds are potent antioxidants (Justino et al., 2002; Terao et al., 2001). The intake of quercetin can inhibit oxidation of LDL and prevent the cardiovascular diseases (de Whalley et al., 1990; Hertog et al., 1993; Manach et al., 1998). Moreover, quercetin and its derivatives have strong anti-inflammation activity (Sato et al., 2020; Carullo et al., 2017; Tejada et al., 2017; Li et al. 2016; and Chen et al., 2016). All of these functions can benefit people’s health.

Table 8. The affinity scores of 11 compounds

EXAMPLE 9

Inhibition of 229E human coronavirus by quercetin and quercetin-3-glycoside

Summary:

Quercetin and quercetin-3 -glycoside inhibited the replication of virus in Huh-7 cells. Quercetin showed the most effective inhibition.

Materials and Reagents:

Materials and reagents include virus clarified suspensions, sterile lx PBS buffer, sterile round- or flat-bottom 96-well plates, MDCK-coated 96-well plates, infection media (DMEM, 0.1% BSA, 1 pg/mL Trypsin), multichannel and single-channel pipettes with sterile tips, sterile reagent boats, and incubator. DEME is the abbreviation of the medium sold under the tradename GIBCO™ Dulbecco's Modified Eagle Medium (Life Technologies Corporation, Carlsbad, California, United States of America).

Virus strain:

229E human coronavirus (HuCoV229e)

Human cells:

Huh-7 cells

Experimental:

1. HuCoV229E was grown on Huh-7 cells and add 50 pl of virus + media per sample

2. Incubate 60 min at room temperature

3. Remove media, rinse with PBS, and replace with 100 pL 10% fetal bovine serum (FBS) containing quercetin, or quercetin-3 -glycoside (isoquercitrin) (0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, or 50 μM)

4. Plate 10,000 Huh-7 per well in 96 well plates

5. Four replicates for 50% tissue culture infective dose (TCID50) protocol:

• Seed Huh cells at 1x10 ⁴ per well in 96 well plate, incubate overnight

• Prepare serial 10-fold dilutions in Minimum Essential Medium (MEM) Eagle and add 50 pl per well

• Infect cells for 90 min

• Aspirate supernatants and add 150 pl per well of MEM plus ATB

• Incubate for 6 days, count wells

6. Serial 10-fold dilutions, infect with 50 pl per well for 2 h, add 50 pl DMEM 10%

7. Cytopathic effect (CPE) was examined visually at day 4 post infection by counting structural changes of Huh-7 cells.

The abnormal cells were recorded to show the inhibitory effects of compounds on viral replication in Huh cells. The fewer the abnormal cells there were, the more effect the compound had.

Figure 16 shows inhibition of quercetin on 229E coronavirus replication in Huh-7 cells. Figure 16A shows that, starting with 5.0 μM, quercetin effectively inhibits the replication of 229E human coronavirus, which causes a common flu. Figure 16B shows that the half maximum effective concentration (EC50) of quercetin was 4.88 μM.

Figure 17 shows inhibition of quercetin-3 -glycoside on 229E virus replication in Huh- 70 cells. Starting with 2.5 μM, quercetin-3 -glycoside (isoquercitrin) inhibits the replication of 229E human coronavirus, which causes a common flu. EXAMPLE 10

Inhibition of 229E human coronavirus by green tea, EGCG, epicatechin, and taxifolin

Summary:

Green tea, EGCG, and epicatechin inhibited the replication of virus in Huh-70 cells. Green tea extracts showed a highly effective inhibition.

Materials and Reagents:

Materials and reagents included virus clarified suspensions, sterile lx PBS buffer, sterile round- or flat-bottom 96-well plates, MDCK-coated 96-well plates, infection media (DMEM, 0.1% bovine serum albumin (BSA), 1 pg/mL Trypsin), multichannel and singlechannel pipettes with sterile tips, sterile reagent boats, and incubator. DEME is the abbreviation of a culture medium sold under the tradename of GIBCO™ Dulbecco's Modified Eagle Medium (Life Technologies Corporation, Carlsbad, California, United States of America).

Virus Strain:

229E human coronavirus strain (HuCoV229e)

Human cells:

Huh-7 cells.

Experimental:

1. HuCoV229e was grown in Huh-7 cells and add 50 pl of virus + media per sample

2. Incubate 60 min at room temperature

3. Remove media, rinse with PBS, and replace with 100 pL 10% FBS containing taxifolin, epigallocatechin-3-O-gallate (EGCG), epicatechin (0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, or 50 μM) or green tea extract (1, 2.5, 5, 10 or 20 pg/mL)

4. Plate 10,000 Huh-7 per well in 96 well plates

5. 4 replicates for 50% tissue culture infective dose (TCID50), protocol:

• Seed Vero cells at 1x10 ⁴ per well in 96 well plate, incubate overnight

• Prepare serial 10-fold dilutions in Minimum Essential Medium (MEM) Eagle and add 50 pl per well

• Infect cells for 90 min,

• Aspirate supernatants and add 150 pl per well of MEM plus ATB

• Incubate for 6 days, count wells

6. Serial 10-fold dilutions, infect with 50 pl per well for 2 h, add 50 pl DMEM 10%

7. Cytopathic effect (CPE) was examined visually at day 4 post infection by counting structural changes of Huh-7 cells. The abnormal cells were recorded to show the inhibitory effects of compounds on viral replication in Huh-7 cells. The fewer the abnormal cells were, the more effective the compound.

The resulting data showed that green tea, EGCG, and epicatechin inhibited the replication of coronavirus in Huh-7 cells. See Figures 18-21. Green tea extracts showed a highly effective inhibition.

REFERENCES

All references listed in the instant disclosure and in the Appendices attached hereto, including but not limited to all patents, patent applications and publications thereof, scientific journal articles, and database entries (including but not limited to GENBANK® biosequence database entries and including all annotations available therein) are incorporated herein by reference in their entireties to the extent that they supplement, explain, provide a background for, and/or teach methodology, techniques, and/or compositions employed herein. The discussion of the references is intended merely to summarize the assertions made by their authors. No admission is made that any reference (or a portion of any reference) is relevant prior art. The right to challenge the accuracy and pertinence of any cited reference is expressly reserved.

Akagi T, Ikegami A, Suzuki Y, Yoshida J, Yamada M, Sato A, Yonemori K (2009) Expression balances of structural genes in shikimate and flavonoid biosynthesis cause a difference in proanthocyanidin accumulation in persimmon (Diospyros kaki Thunb.) fruit. Planta 230 (5):899-915. doi:10.1007/s00425-009-0991-6

Bull-Otterson L, Gray EB, Budnitz DS, Strosnider HM, Schieber LZ, Courtney J, Garcia MC, Brooks JT, Kenzie WRM, Gundlapalli AV (2020) Hydroxychloroquine and Chloroquine Prescribing Patterns by Provider Specialty Following Initial Reports of Potential Benefit for COVID-19 Treatment — United States, January-June 2020. Morbidity and Mortality Weekly Report 69 (35): 1210—1215.

Burak C., Brull V, Langguth P, Zimmermann BF, Stoffel -Wagner B, Sausen U, Stehle P, Wolffram S, Egert S (2017) Higher plasma quercetin levels following oral administration of an onion skin extract compared with pure quercetin dihydrate in humans. Eur. J. Nutr. 56 (1)343 -353.

Cao B, Wang Y, Wen D, Liu W, Wang J, Fan G, Ruan L, Song B, Cai Y, Wei M, Li X, Xia J, Chen N, Xiang J, Yu T, Bai T, Xie X, Zhang L, Li C, Yuan Y, Chen H, Li H, Huang H, Tu S, Gong F, Liu Y, Wei Y, Dong C, Zhou F, Gu X, Xu J, Liu Z, Zhang Y, Li H, Shang L, Wang K, Li K, Zhou X, Dong X, Qu Z, Lu S, Hu X, Ruan S, Luo S, Wu J, Peng L, Cheng F, Pan L, Zou J, Jia C, Wang J, Liu X, Wang S, Wu X, Ge Q, He J, Zhan H, Qiu F, Guo L, Huang C, Jaki T, Hayden FG, Horby PW, Zhang D, Wang C (2020) A Trial of Lopinavir-Ritonavir in Adults Hospitalized with Severe Covid- 19. N Engl J Med 382 (19): 1787- 1799. doi: 10.1056/NEJMoa2001282.

Careri M, Corradini C, Elviri L., Nicoletti I, Zagnoni I (2003) Direct HPLC analysis of quercetin and trans-resveratrol in red wine, grape, and winemaking byproducts. J Agnc Food Chem 51(18): 5226-5231.

Carullo G, Cappello AR, Frattaruolo L, Badolato M, Armentano B, Aiello F (2017) Quercetin and derivatives: useful tools in inflammation and pain management. Future Med. Chem. 9 (1): 79-93.

CDC. FDA, (2021) Allergic Reactions Including Anaphylaxis After Receipt of the First Dose of Modema COVID-19 Vaccine — United States, December 21, 2020-January 10, 2021. Morbidity and Mortality Weekly Report, 70(4): 125-129.

Chavarria-Mir6 G, Anfruns-Estrada E, Guix S, Paraira M, Galofre B, Saanchez G, Pinto R, Bosch A (2020) Sentinel surveillance of SARS-CoV-2 in wastewater anticipates the occurrence of COVID-19 cases. medRxiv:2020.2006.2013.20129627. doi: 10.1101/2020.06.13.20129627.

Chen L, Gui C, Luo X, Yang Q, Gunther S, Scandella E, Drosten C, Bai D, He X, Ludewig B, Chen J, Luo H, Y ang Y, Y ang Y, Zou J, Thiel V, Chen K, Shen J, Shen X, Jiang H (2005) Cinanserin Is an Inhibitor of the 3C-Like Proteinase of Severe Acute Respiratory Syndrome Coronavirus and Strongly Reduces Virus Replication In Vitro. Journal ofVirology 79 (11):7095-7103. doi:10.1128/jvi.79.11.7095-7103.2005.

Chen S, Jiang HM, Wu XS, Fang J. (2016) Therapeutic Effects of Quercetin on Inflammation, Obesity, and Type 2 Diabetes. Mediat. Inflamm. 2016:9340637.

Chen Y, Liu Q, Guo D (2020) Emerging coronaviruses: Genome structure, replication, and pathogenesis. J Med Virol 92(4):418-423.

Cohen J (2021) South Africa suspends use of AstraZeneca’s COVID- 19 vaccine after it fails to clearly stop virus variant. Science. doi:10.1126/science.abg9559.

Dai W, Zhang B, Jiang X-M, Su H, Li J, Zhao Y, Xie X, Jin Z, Peng J, Liu F, Li C, Li Y, Bai F, Wang H, Cheng X, Cen X, Hu S, Yang X, Wang J, Liu X, Xiao G, Jiang H, Rao Z, Zhang L-K, Xu Y, Yang H, Liu H (2020a) Structure-based design of antiviral drug candidates targeting the SARS-CoV-2 main protease. Science 368 (6497): 1331-1335. doi: 10.1126/science.abb4489.

Dai X, Liu Y, Zhuang J, Yao S, Liu L, Jiang X, Zhou K, Wang Y, Xie D-Y, Bennetzen JL, Gao L, Xia T (2020b) Discovery and characterization of tannase genes in plants: roles in hydrolysis of tannins. New Phytol 226(4): 1104-1116. Day AJ, Williamson G (2001) Biomarkers for exposure to dietary flavonoids: a review of the current evidence for identification of quercetin glycosides in plasma. Br. J. Nutr. 86: S105-S110.

De Bruyne T, Pieters L, Witvrouw M, De Clercq E, Vanden Berghe D, Vlietinck AJ (1999) Biological evaluation of proanthocyanidin dimers and related polyphenols. J Nat Prod 62 (7):954-958. de Whalley CV, Rankin SM, Hoult JR, Jessup W, Leake DS (1990) Flavonoids inhibit the oxidative modification of low density lipoproteins by macrophages. Biochem Pharmacol 39(11): 1743-50.

Ding Q, Lu P, Fan Y, Xia Y, Liu, M (2020)The clinical characteristics of pneumonia patients coinfected with 2019 novel coronavirus and influenza virus in Wuhan, China. J Med Virol 92(9): 1549-1555.

Dooling K, Marin M, Wallace M, McClung N, Chamberland M, Lee GM, Talbot HK, Romero JR, Bell BP, Oliver SE (2021) The Advisory Committee on Immunization Practices’ Updated Interim Recommendation for Allocation of COVID-19 Vaccine — United States, December 2020. Morbidity and Mortality Weekly Report 69 (5152): 1657- 1660.

Egert S, Wolffram S, Schulze B, Langguth P, Hubbermann EM, Schwarz K,Adolphi B, Bosy- Westphal A, Rimbach G, Muller MJ (2012) Enriched cereal bars are more effective in increasing plasma quercetin compared with quercetin from powder-fdled hard capsules. Br. J. Nutr. 107(4):539-546.

Erlund I, Silaste ML, Alfthan G, Rantala M, Kesaniemi YA, Aro A (2002) Plasma concentrations of the flavonoids hesperetin, naringenin and quercetin in human subjects following their habitual diets, and diets high or low in fruit and vegetables. Eur. J. Clin. Nutr. 56(9):891-898.

Fischer TC, Mirbeth B, Rentsch J, Sutter C, Ring L, Flachowsky H, Habegger R, Hoffmann T, Hanke MV, Schwab W (2014) Premature and ectopic anthocyanin formation by silencing of anthocyanidin reductase in strawberry (Fragaria x ananassa). New Phytol 201 (2):440-451. doi:10.1111/nph.l2528.

Foo LY, Lu Y, Howell AB, Vorsa N (2000a) The structure of cranberry proanthocyanidins which inhibit adherence of uropathogenic P-fimbriated Escherichia coli in vitro. Phytochemistry 54:173-181.

Foo LY, Lu Y, Howell AB, Vorsa N (2000b) A-type proanthocyanidin trimers from cranberry that inhibit adherence of uropathogenic P-fimbriated Escherichia coli. J Nat Prod 63:1225-1228. Fossen T, Rayyan S, Andersen OM (2004) Dimeric anthocyanins from strawberry (Fragaria ananas sa) consisting of pelargonidin 3 -glucoside covalently linked to four flavan-3- ols. Phytochemistry 65 (10): 1421-1428. doi:10.1016/j.phytochem.2004.05.003.

Fowler ZL, Koffas MA (2009) Biosynthesis and biotechnological production of flavanones: current state and perspectives. Appl Microbiol Biotechnol 83(5):799-808.

Gharpure R, Guo A, Bishnoi CK, Patel U, Gifford D, Tippins A, Jaffe A, Shulman E, Stone N, Mungai E, Bagchi S, Bell J, Srinivasan A, Patel A, Link-Gelles R (2021) Early COVID- 19 First-Dose Vaccination Coverage Among Residents and Staff Members of Skilled Nursing Facilities Participating in the Pharmacy Partnership for Long-Term Care Program — United States, December 2020-January 2021. Morbidity and Mortality Weekly Report, 70(5): 178-182.

Gu L, Keim M, Hammerstone JF, Beecher G, Cunningham D, AVAnnozzi S, Prior RL (2002) Fractionation of polymeric procyanidins from lowbush blueberry and quantification of procyanidins in selected foods with an optimized normal-phase HPLC-MS fluorescent detection method. J Agric Food Chem 50:4852-4860.

Helms J, Kremer S, Merdji H, Clere-Jehl R, Schenck M, Kummerlen C, Collange O, Boulay C, Fafi-Kremer S, Ohana M, Anheim M, Meziani F (2020) Neurologic Features in Severe SARS-CoV-2 Infection. New England Journal of Medicine 382 (23):2268- 2270. doi: 10.1056/NEJMc2008597.

Hertog MG, Feskens EJ, Hollman PC, Katan MB, Kromhout D (1993) Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study. Lancet 342 (8878): 1007-11.

Holshue ML, DeBolt C, Lindquist S, Lofy KH, Wiesman J, Bruce H, Spitters C, Ericson K, Wilkerson S, Tural A, Diaz G, Cohn A, Fox L, Patel A, Gerber S1, Kim L. Tong S, Lu X, Lindstrom S, Pallansch MA, Weldon WC, Biggs HM, Uyeki TM, Pillai SK, (2020) First Case of 2019 Novel Coronavirus in the United States. N Engl J Med 382 (10):929-936.

Hostetler GL, Ralston RA, Schwartz SJ, (2017) Flavones: Food Sources, Bioavailability, Metabolism, and Bioactivity. Adv Nutr 8(3):423-435.

Howell AB, Reed JD, Krueger CG, Winterbottom R, Cunningham DG, Leahy M (2005) A- type cranberry proanthocyanidins and uropathogenic bacterial anti-adhesion activity. Phytochemistry 66(18):2281-2291.

Huang HH, Liao D, Dong Y, Pu R (2020) Effect of quercetin supplementation on plasma lipid profiles, blood pressure, and glucose levels: a systematic review and meta-analysis. Nutr. Rev. 78(8):615-626. Hussain S, Pan J, Chen Y, Yang Y, Xu J, Peng Y, Wu Y, Li Z, Zhu Y, Tien P, Guo D (2005) Identification of novel subgenomic RNAs and noncanonical transcription initiation signals of severe acute respiratory syndrome coronavirus. J Virol, 79(9):5288-95. lacopini P, Baldi M, Storchi P, Sebastiani L (2008) Catechin, epicatechin, quercetin, rutin and resveratrol in red grape: Content, in vitro antioxidant activity and interactions. J Food Compos Anal 21(8):589-598

Iwasawa A, Niwano Y, Mokudai T, Kohno M (2009) Antiviral activity of proanthocyanidin against feline calicivirus used as a surrogate for noroviruses, and coxsackievirus used as a representative enteric virus. Biocontrol Sci 14(3): 107-111. doi:10.4265/bio,14.107

Jin Z, Du X, Xu Y, Deng Y, Liu M, Zhao Y, Zhang B, Li X, Zhang L, Duan Y, Yu J, Wang L, Yang K, Liu F, You T, Liu X, Yang X, Bai F, Liu H, Liu X, Guddat LW, Xiao G, Qin C, Shi Z, Jiang H, Rao Z, Yang H (2020a) Structure-based drug design, virtual screening and high-throughput screening rapidly identify antiviral leads targeting COVID-19. bioRxiv:2020.2002.2026.964882. doi: 10.1101/2020.02.26.964882

Jin Z, Du X, Xu Y, Deng Y, Liu M, Zhao Y, Zhang B, Li X, Zhang L, Peng C, Duan Y, Yu J, Wang L, Yang K, Liu F, Jiang R, Yang X, You T, Liu X, Yang X, Bai F, Liu H, Liu X, Guddat LW, Xu W, Xiao G, Qin C, Shi Z, Jiang H, Rao Z, Yang H (2020b) Structure of M ^pro from COVID- 19 virus and discovery of its inhibitors. bioRxiv:2020.2002.2026.964882. doi: 10.1101/2020.02.26.964882

Jin Z, Zhao Y, Sun Y, Zhang B, Wang H, Wu Y, Zhu Y, Zhu C, Hu T, Du X, Duan Y, Yu J, Yang X, Yang X, Yang K, Liu X, Guddat LW, Xiao G, Zhang L, Yang H, Rao Z (2020c) Structural basis for the inhibition of SARS-CoV-2 main protease by antineoplastic drug carmofur. Nature Structural & Molecular Biology 27(6): 529-532. doi: 10.1038/s41594-020-0440-6

Jin Z, Du, X, Xu Y, Deng Y, Liu M, Zhao Y, Zhang B, Li X, Zhang L, Peng C, Duan Y, Yu J, Wang L, Yang K, Liu F, Jiang R, Yang X, You T, Liu X, Yang X, Bai F, Liu H, Liu X, Guddat LW, Xu W, Xiao G, Qin C, Shi Z, Jiang H, Rao Z, Yang H (2020d) Structure of M(pro) from SARS-CoV-2 and discovery of its inhibitors. Nature 582(7811):289-293.

Justino G, Santos M, Mira L (2002) Plasma metabolites of quercetin and their antioxidant potential. Free Radic. Biol. Med. 33:S203-S203.

Kenilworth NJ (2021) Merck Discontinues Development of SARS-CoV2/COVID-l 9 Vaccine Candidates; Continues Development of Two Investigational Therapeutic Candidates. BUS1NESS WIRE, January 25, 2021. Khan SU, Htar T-T (2020) Deciphering the binding mechanism of Dexamethasone against SARS-CoV-2 Main Protease: Computational molecular modelling approach. Chemrxivorg. doi : 10.26434/ chemrxiv.12517535. vl.

Kim Y, Liu H, Galasiti Kankanamalage AC, Weerasekara S, Hua DH, Groutas WC, Chang KO, Pedersen NC (2016) Reversal of the Progression of Fatal Coronavirus Infection in Cats by a Broad-Spectrum Coronavirus Protease Inhibitor. PLoS Pathog 12(3):el005531.

Kim JH, Marks F, Clemens JD (2021) Looking beyond COVID- 19 vaccine phase 3 trials. Nature Medicine 27:205-211.

Knoll MD, Wonodi C (2021) Oxford-AstraZeneca COVID-19 vaccine efficacy. The Lancet 397(10269):72-74.

Larners MM, Beumer J, van der Vaart J, Knoops K, Puschhof J, Breugem TI, Ravelli RBG, Paul van Schayck J, Mykytyn AZ, Duimel HQ, van Donselaar E, Riesebosch S, Kuijpers HJH, Schipper D, van de Wetering WJ, de Graaf M, Koopmans M, Cuppen E, Peters PJ, Haagmans BL, Clevers H (2020) SARS-CoV-2 productively infects human gut enterocytes. Science 369(6499):50-54. doi:10.1126/science.abcl669.

Levites Y, Amit T, Mandel S, Youdim MBH (2003) Neuroprotection and neurorescue against Aβtoxicity and PKC-dependent release of non-amyloidogenic soluble precursor protein by green tea polyphenol (-)- epigallocatechin-3 -gallate. Faseb J 17:952-954. doi:10.1096/fj.02-0881fje.

Li M-H, Jang J-H, Sun B, Surh Y-J (2004) Protective effects of oligomers of grape seed polyphenols against beta-amyloid-induced oxidative cell death. Ann NY Acad Sci 1030(l):317-329. doi: 10.1196/annals.1329.040.

Li Y, Yao JY, Han CY, Yang JX, Chaudhry MT, Wang SN, Liu HN, Yin YL (2016) Quercetin, Inflammation and Immunity. Nutrients 8(3): 14.

Liu YJ, Gao LP, Liu L, Yang Q, Lu ZW, Nie ZY, Wang YS, Xia T (2012) Purification and Characterization of a Novel Galloyltransferase Involved in Catechin Galloylation in the Tea Plant (Camellia sinensis). J Biol Chem 287(53):44406-44417. doi: 10.1074/jbc.Ml 12.403071.

Loke WM, Hodgson JM, Proudfoot JM, McKinley AJ, Puddey IB, Croft KD (2008) Pure dietary flavonoids quercetin and (-)-epicatechin augment nitric oxide products and reduce endothelin-1 acutely in healthy men. Am J Clin Nutr 88(4): 1018-1025.

Lopez-Serrano M, Barcelo AR (1997) Kinetic properties of (+)-catechin oxidation by a basic peroxidase ioenzyme from strawberries. J Food Sci 62(4):676-723. doi: doi: 10.1111/j .1365-2621.1997. tb 15433.x. MacRae K, Connolly K, Vella R, Penning A (2019) Epicatechin's cardiovascular protective effects are mediated via opioid receptors and nitric oxide. Eur J Nutr 58(2): 515-527. doi:10.1007/s00394-018-1650-0.

Manach C, Morand C, Crespy V, Demigne C, Texier O, Regerat F, Remesy C (1998) Quercetin is recovered in human plasma as conjugated derivatives which retain antioxidant properties. FEBS Lett. 426(3):331 -336.

McCarthy KR, Rennick LJ, Nambulli S, Robinson-McCarthy LR, Bain WG, Haidar G, Duprex WP (2020) Natural deletions in the SARS-CoV-2 spike glycoprotein drive antibody escape. bioRxiv, 2020.11.19.389916.

Meng XF, Maliakal P, Lu H, Lee MJ, Y ang CS (2004) Urinary and plasma levels of resveratrol and quercetin in humans, mice, and rats after ingestion of pure compounds and grape juice. J. Agric. Food Chem. 52(4):935-942.

Miller KB, Hurst WJ, Flannigan N, Ou BX, Lee CY, Smith N, Stuart DA (2009) Survey of Commercially Available Chocolate- and Cocoa-Containing Products in the United States. 2. Comparison of Flavan-3-ol Content with Nonfat Cocoa Solids, Total Polyphenols, and Percent Cacao. J Agric Food Chem 57(19):9169-9180. doi:10.1021/jf901821x.

Mohammadi-Sartang M, Mazloom Z, Sherafatmanesh S, Ghorbani M, Firoozi D (2017) Effects of supplementation with quercetin on plasma C-reactive protein concentrations: a systematic review and meta-analysis of randomized controlled trials. Eur. J. Clin. Nutr. 71(9): 1033-1039.

Molan AL, Attwood GT, Min BR, McNabb WC (2001) The effect of condensed tannins from Lotus pedunculatus and Lotus comiculatus on the growth of proteolytic rumen bacteria in vitro and their possible mode of action. Can J Microbiol 47(7):626-633. doi: 10.1139/cjm-47-7-626

Monagas M, Gomez CC, Bartolome B, Laureano O, da Ricardo SJM (2003) Monomeric, oligomeric, and polymeric flavan-3-ol composition of wines and grapes from Vitis vinifera L. cv. Graciano, Tempranillo, and Cabernet Sauvignon. J Agric Food Chem 51:6475-6481.

Murphy KJ, Chronopoulos AK, Singh I, Francis MA, Moriarty H, Pike MJ, Turner AH, Mann NJ, Sinclair AJ (2003) Dietary flavanols and procyanidin oligomers from cocoa (Theobroma cacao) inhibit platelet function. Am J Clin Nutr 77(6): 1466-1473.

OhataM, Koyama Y, Suzuki T, Hayakawa S, Saeki K, Nakamura Y, IsemuraM (2005) Effects of tea constituents on cell cycle progression of human leukemia U937 cells. Biomed Res 26(1): 1-7. Olthof MR, Hollman PC, Vree TB, Katan MB (2000) Bioavailabilities of quercetin-3- glucoside and quercetin-4'-glucoside do not differ in humans. J Nutr 130(5): 1200-3.

Painter EM, Ussery EN, Patel A, Hughes MM, Zell ER, Moulia DL, Scharf LG, Lynch M, Ritchey MD, Toblin RL, Murthy BP, Harris LQ, Wasley A, Rose DA, Cohn A, Messonnier NE (2021) Demographic Characteristics of Persons Vaccinated During the First Month of the COVID- 19 Vaccination Program — United States, December 14, 2020-January 14, 2021. Morbidity and Mortality Weekly Report 70(5): 174-177.

Panneerselvam M, Tsutsumi YM, Bonds JA, Horikawa YT, Saldana M, Dalton ND, Head BP, Patel PM, Roth DM, Patel HH (2010) Dark chocolate receptors: epicatechin-induced cardiac protection is dependent on delta-opioid receptor stimulation. Am J Physiol- Heart Circul Physiol 299(5):H1604-H1609. doi: 10.1152/ajpheart.00073.2010.

Pillaiyar T, Manickam M, Namasivayam V, Hayashi Y, Jung S-H (2016) An Overview of Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) 3 CL Protease Inhibitors: Peptidomimetics and Small Molecule Chemotherapy. J Med Chem 59(14):6595-6628. doi:10.1021/acs.jmedchem.5b01461.

Ramajayam R, Tan KP, Liang PH (2011) Recent development of 3C and 3CL protease inhibitors for anti-coronavirus and anti-picomavirus drug discovery. Biochem Soc Trans 39(5): 1371-5.

Ren Z, Yan L, Zhang N, Guo Y, Yang C, Lou Z, Rao Z (2013) The newly emerged SARS- like coronavirus HCoV-EMC also has an "Achilles' heel": current effective inhibitor targeting a 3C-like protease. Protein Cell 4(4):248-50.

Rousserie P, Rabot A, Geny-Denis L (2019) From Flavanols Biosynthesis to Wine Tannins: What Place for Grape Seeds? J Agric Food Chem 67(5): 1325-1343. doi: 10.1021/acs.jafc.8b05768.

Sanders JM, Monogue ML, Jodlowski TZ, Cutrell JB (2020) Pharmacologic Treatments for Coronavirus Disease 2019 (COVID-19): A Review. JAMA 323(18): 1824-1836. doi: 10.1001/jama.2020.6019.

Sato S, Mukai Y (2020) Modulation of chronic inflammation by quercetin: The beneficial effects on obesity. J. Inflamm. Res.13:421-431.

Schewe T, Kuhn H, Sies H (2002) Flavonoids of Cocoa Inhibit Recombinant Human 5- Lipoxygenase. J Nutr 132(7): 1825-1829.

Schewe T, Sadik C, Klotz LO, Yoshimoto T, Kuhn H, Sies H (2001) Polyphenols of cocoa: inhibition of mammalian 15 -lipoxygenase. Biol Chem 382(12):1687-1696.

Selvaraj V, Dapaah-Afriyie K, Finn A, Flanigan TP (2020) Short-Term Dexamethasone in Sars-CoV-2 Patients. Rhode Island Medical Journal 103:39-43. Serafini M, Bugianesi R, Maiani G, Valtuena S, De Santis S, Crozier A (2003) Plasma antioxidants from chocolate. Nature 424: 1013.

Shi YL, Williamson G (2016) Quercetin lowers plasma uric acid in pre-hyperuricaemic males: a randomised, double-blinded, placebo-controlled, cross-over trial. Br. J. Nutr. 115(5): 800-806.

Snyder SM, Zhao BX, Luo T, Kaiser C, Cavender G, Hamilton-Reeves J, Sullivan DK, Shay NF 2016) Consumption of Quercetin and Quercetin-Containing Apple and Cherry Extracts Affects B1ood Glucose Concentration, Hepatic Metabolism, and Gene Expression Patterns in Obese C57BL/6J High Fat-Fed Mice. J. Nutr. 146(5): 1001- 1007.

Suganuma M, Saha A, Fujiki H (2011) New cancer treatment strategy using combination of green tea catechins and anticancer drugs. Cancer Science 102(2):317-323. doi: 10.1111/j.1349-7006.2010.01805.X.

Takahashi T, Kamiya T, Hasegawa A, Yokoo Y (1999) Procyanidin oligomers selectively and intensively promote proliferation of mouse hair epithelial cells in vitro and activate hair follicle growth in vivo. J Invest Dermatol 112(3): 310-316.

Tejada S, Nabavi SM, Capo X, Martorell M, Bibiloni MD, Tur JA, Pons A, Sureda A (2017) Quercetin effects on exercise induced oxidative stress and inflammation. Curr. Org. Chem. 21(4):348-356.

Terao J, Yamaguchi S, Shirai M, Miyoshi M, Moon JH, Oshima S, Inakuma T, Tsushida T, Kato Y, (2001) Protection by quercetin and quercetin 3-O-beta-glueuronide of peroxynitrite-induced antioxidant consumption in human plasma low-density lipoprotein. Free Radic. Res. 35(6):925-931. van Doremalen N, Bushmaker T, Morris DH, Holbrook MG, Gamble A, Williamson BN, Tamin A, Harcourt JL, Thornburg NJ, Gerber S1, Lloyd-Smith JO, de Wit E, Munster VJ (2020) Aerosol and Surface Stability of SARS-CoV-2 as Compared with SARS- CoV-1. New England Journal of Medicine 382(16): 1564-1567. doi:10.1056/NEJMc2004973.

Wang C, Horby PW, Hayden FG, Gao GF (2020a) A novel coronavirus outbreak of global health concern. Lancet (London, England) 395(10223):470-473. doi:10.1016/S0140- 6736(20)30185-9.

Wang M, Cao R, Zhang L, Yang X, Liu J, Xu M, Shi Z, Hu Z, Zhong W, Xiao G (2020b) Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Res 30(3):269-271. doi: 10.1038/s41422-020-0282-0. Wang P, Liu Y, Zhang L, Wang W, Hou H, Zhao Y, Jiang X, Yu J, Tan H, Wang Y, Xie D- Y, Gao L, Xia T (2020c) Functional demonstration of plant flavonoid carbocations proposed to be involved in the biosynthesis of proanthocyanidins. The Plant Journal 101(1): 18-36. doi: 10.1111/tpj.14515.

Weinreb O, Mandel S, Amit T, Youdim MBH (2004) Neurological mechanisms of green tea polyphenols in Alzheimer's and Parkinson's diseases. J Nutriti Biochem 15(9): 506- 516.

WHO (2020a) Statement on the second meeting of the International Health Regulations (2005) Emergency Committee regarding the outbreak of novel coronavirus (2019-nCoV). World Health Organization, Geneva, Switzerland.

WHO (2020b) WHO Director-General's opening remarks at the media briefing on COVID- 19 - 11 March 2020. World Health Organization, Geneva, Switzerland.

Wu Y-C, Chen C-S, Chan Y-J (2020) The outbreak of COVID-19: An overview. J Chin Med Assoc 83(3):217-220. doi: 10.1097/jcma.0000000000000270.

Xiao F, Tang M, Zheng X, Liu Y, Li X, Shan H (2020) Evidence for Gastrointestinal Infection of SARS-CoV-2. Gastroenterology 158(6): 1831 -1833 el 833. doi: 10.1053/j.gastro.2020.02.055.

Xie D-Y, Dixon RA (2005) Proanthocyanidin biosynthesis - still more questions than answers? Phytochemistry 66(18):2127-2144.

Xie et al. (2006) Plant J 45, 895-907.

Xue X, Yu H, Yang H, Xue F, Wu Z, Shen W, Li J, Zhou Z, Ding Y, Zhao Q, Zhang XC, Liao M, Bartlam M, Rao Z (2008) Structures of two coronavirus main proteases: Implications for substrate binding and antiviral drug design. Journal of Virology 82 (5):2515-2527. doi: 10.1128/jvi.O2114-07.

Y ang H, Xie W, Xue X, Y ang K, Ma J, Liang W, Zhao Q, Zhou Z, Pei D, Ziebuhr J, Hilgenfeld R, Yuen KY, Wong L, Gao G, Chen S, Chen Z, Ma D, Bartlam M, Rao Z (2005) Design of wide-spectrum inhibitors targeting coronavirus main proteases. PLoS Biol 3(10):e324.

Ye G, Wang X, Tong X, Shi Y, Fu ZF, Peng G (2020) Structural Basis for Inhibiting Porcine Epidemic Diarrhea Virus Replication with the 3C-Like Protease Inhibitor GC376. Viruses 12(2):240.

Yuzuak S, Ballington J, Xie DY (2018) HPLC-qTOE-MS/MS-Based Profiling of Flavan-3- ols and Dimeric Proanthocyanidins in Berries of Two Muscadine Grape Hybrids FLH 13-11 and FLH 17-66. Metabolites 8(4):57. doi: 10.3390/metabo8040057. Zhang L, Lin D, Sun X, Curth U, Drosten C, Sauerhering L, Becker S, Rox K, Hilgenfeld R (2020a) Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved a-ketoamide inhibitors. Science 368(6489):409-412. doi: 10.1126/science.abb3405.

Zhang L, Liu Y (2020b) Potential interventions for novel coronavirus in China: A systematic review. J Med Virol 92(5):479-490.

Zhao L, Jiang XL, Qian YM, Wang PQ, Xie DY, Gao LP, Xia T (2017) Metabolic Characterization of the Anthocyanidin Reductase Pathway Involved in the Biosynthesis of Flavan-3-ols in Elite Shuchazao Tea (Camellia sinensis) Cultivar in the Field. Molecules 22(12):21. doi:10.3390/molecules22122241.

Zhu Y, Peng QZ, Li KG, Xie DY (2014) Molecular cloning and functional characterization of the anthocyanidin reductase gene from Vitis bellula. Planta 240(2):381-398. doi: 10.1007/s00425-014-2094-2.

Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, Zhao X, Huang B, Shi W, Lu R, Niu P, Zhan F, Ma X, Wang D, Xu W, Wu G, Gao GF, Tan W (2020a) China Novel Coronavirus, I.; Research, T., A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med, 382(8):727-733.

Zhu Y, Xie D-Y (2020b) Docking characterization and in vitro inhibitory activity of flavan-3- ols and dimeric proanthocyanidins against the main protease activity of SARS-Cov-2. Front. Plant Sci. 11:601316.

It will be understood that various details of the presently disclosed subject matter may be changed without departing from the scope of the presently disclosed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.