Table 2.

Studies screening plant-derived NPs for SARS-CoV-2 S-ACE2 inhibitory activity.

Authors	Study design	Natural product	Analytical techniques	Summary of major findings
Al-Shuhaib et al. (2022)	In silico	Iraqi medicinal plants	Molecular docking, molecular dynamics	Epicatechin from Hypericum perforatum displayed high affinity for ACE2, interacting strongly and stably with residues directly involved in interaction with S RBD.
Campos et al. (2023)	In vitro	Bark extracts of Ampelozizyphus amazonicus	LC-MS, antibody-based inhibitor screening kit, infectivity assay	Aqueous and ethanol extracts inhibited the formation of the S-ACE2 complex by at least 50% and inhibited SARS-CoV-2 replication in vitro. The most promising EC50 values of extracts were <25 µg/ml. Phytochemical analysis identified a range of bioactive compounds including saponins, triterpenes, and phenolic compounds.
Chen C. et al. (2022)	In silico	Bioactive cannabinoids	Molecular docking, molecular dynamics	Luteolin, cannabigerovarinic acid (CBVGA), and cannabinolic acid (CBNA) had the highest affinity for the S-ACE2 complex out of the 42 cannabinoids screened. Based on interactions with key residues of the S RBD, luteolin and CBNA may inhibit SARS-CoV-2 entry.
Chen GY. et al. (2022)	In vitro and in silico	Database of extracts and compounds	Isothermal titration calorimetry (ITC), molecular docking, molecular dynamics, pseudovirus assay	39 compounds with affinity for S RBD were identified. Of these compounds, 10 µM dioscin, celastrol, epimedin C, amentoflavone, torvoside K, and saikosaponin C consistently inhibited 50-90% of spike pseudovirus entry into 293T-ACE2 cells.
El Hawary et al. (2020)	In silico	Phytocompounds from Tecoma spp.	LC-MS, molecular docking	12 phytocompounds isolated from eight Tecoma species showed moderate to strong binding affinity with the S-ACE2 interface. Succinic acid displayed the strongest affinity (-6.77 kcal/mol).
Gao et al. (2023)	In vitro	Japanese honeysuckle (Lonicera japonica)	LC-MS, antibody-based inhibitor screening kit, ACE2 activity assay,	Water and ethanol extracts inhibited the S-ACE2 interaction (65%, 100%) and ACE2 activity (90%, 62%). 36 bioactive compounds identified in extracts including flavonoids quercetin and luteolin as well as 10 novel compounds.
Goc et al. (2021b)	In vitro	Polyphenols and plant extracts	Antibody-based inhibitor screening kit, pseudovirus assay, cell fusion assay, TMPRSS2, CatL, and ACE2 activity assays, ACE2 binding assay, endosomal/lysosomal pH assay	Brazilin, theaflavin-3,3′-digallate (TF-3), and curcumin had strong affinity for S RBD, blocked pseudovirus fusion and entry, and reduced TMPRSS2 activity. TF-3 and curcumin also decreased ACE2 and CatL activity. TF-3 also decreased CatL expression by increasing endosomal pH.
González-Maldonado et al. (2022)	In vitro	Plant and fungal extracts, essential oils	Pseudovirus assay	Stachytarpheta cayennensis extract reduced D614G spike pseudovirus infectivity with an IC50 of 91.65 µg/ml. β-caryophyllene and Phoradendron liga extract also significantly decreased pseudovirus infectivity. Essential oils tested displayed no inhibitory effect and some appeared to increase infectivity.
Invernizzi et al. (2022)	In vitro	Gunnera perpensa	Bead-based protein binding assay (AlphaScreen), LC-MS	G. perpensa extract inhibited the S-ACE2 interaction with an IC50 of <0.001 µg/ml. Chromatographical techniques identified the ellagitannins punicalin and punicalagin as major constituents of G. perpensa. These isolated compounds inhibited the S-ACE2 interaction with respective IC50 values of 9 nM and 29 nM.
Kim et al. (2021)	In vitro and in silico	Geraniin	Antibody-based inhibitor screening kit, biolayer interferometry (BLI), molecular docking, molecular dynamics	Geraniin inhibited the S-ACE2 interaction (IC50 = 4.2 µM) and displayed strong affinity for both S RBD and ACE2 through interactions with multiple residues, although affinity for S RBD was higher.
Kumar et al. (2023)	In silico	Flavonoids	Molecular docking, molecular dynamics	Of the 40 flavonoids tested against the Omicron S RBD, five (tomentin A, tomentin C, hyperoside, catechin gallate, and corylifol A) displayed favorable properties including strong binding affinity, stable interaction dynamics, and favorable free binding energies.
Li et al. (2021)	In vitro and in silico	Glycyrrhizic acid	Pseudovirus assay, biotin binding assay, surface plasmon resonance (SPR) analysis, molecular docking	Glycyrrhizic acid (GA) inhibited the S-ACE2 interaction and pseudovirus entry. Pre-treating pseudovirus with GA had a greater effect than pre-treating cells, suggesting preferential binding with S rather than ACE2. Multiple GA binding pockets were identified on S RBD.
Mei et al. (2021)	In vitro and in silico	Chinese ephedra (Ephedra sinica)	Biotin binding assay, SPR analysis, LC-MS, molecular docking, pseudovirus assay	Ephedra sinica extract (ESE) inhibited the S-ACE2 interaction with an IC50 of 95.01 µg/ml and promoted virus-host dissociation. Quinoline-2-carboxylic acids were identified as major chemical constituents of ESE and could block S-ACE2 independently with micromolar IC50 values by interacting with residues in S RBD. These compounds also inhibited pseudovirus entry into multiple cell lines.
Meng et al. (2023)	In vitro and in silico	Flavonoids	Pseudovirus assay, BLI, molecular docking	24 of 31 tested flavonoids inhibited pseudovirus entry by binding to S RBD and blocking the S-ACE2 interaction, with myricetin having the most potent activity (IC50 = 10.27 ± 2.32 µM) and the highest affinity (KD = 9.62 ± 2.11 µM). Molecular docking determined that flavonoids could interact with a highly conserved pocket in the S RBD, highlighting their potential for pan-variant entry inhibition.
Mhatre et al. (2021)	In silico	Catechins	Molecular docking, molecular dynamics	All tested catechins bound strongly to the S protein of wild-type and Alpha E484K variants. Epigallocatechin gallate (EGCG) had the strongest affinity for and the most stable interaction with both variants.
Mondal et al. (2021)	In silico	Biflavone-based antioxidants	Molecular docking	The natural anti-HIV agents hinokiflavone and robustaflavone displayed strong binding affinity for the S2 subunit of the S protein, interacting strongly with the HR1 and HR2 regions, suggesting potential for the inhibition of viral membrane fusion.
Nag et al. (2022)	In silico	Curcumin and other phytochemicals	Molecular docking, molecular dynamics	Curcumin interacted strongly and stably with multiple mutated residues in Omicron S RBD and S-ACE2 complex. The interaction also induced structural changes in S, possibly contributing to reduced affinity for ACE2.
Ohishi et al. (2022)	In vitro and in silico	Phytochemicals	ELISA, molecular docking, infectivity assay	Of the 10 phytochemicals tested, EGCG had the highest inhibitory activity against the S-ACE2 interaction (93.3%, 100 µM, IC50 = 33.9 µM). Curcumin also showed moderate activity (67%, 100 µM). EGCG interacted strongly with residues at the S-ACE2 interface and prevented SARS-CoV-2 infection in vitro.
Pan et al. (2023)	In vitro and in silico	Myricetin	Infectivity assay, molecular docking, BLI, pseudovirus assay, animal studies	Myricetin inhibited the replication of live SARS-CoV-2 (EC50 = 55.18 µM) and human coronavirus HCoV-229E (EC50 = 53.51 µM). The flavonoid also bound strongly to the S RBD of wild-type and mutated SARS-CoV-2, successfully blocking their interaction with ACE2. Pseudovirus entry into cells expressing ACE2 was also inhibited. Furthermore, myricetin exerted multiple anti-inflammatory effects in rat and mouse models.
Risener et al. (2023)	In vitro	Plant and fungal extracts	Pseudovirus assay, infectivity assay, LC-MS	Solidago altissima, Salix nigra, and Pteridium aquilinum extracts inhibited the entry of wild-type, Alpha, Beta, Gamma, and Delta spike pseudoviruses (EC50 <10 µg/ml). S. altissima and P. aquilinum extracts blocked the entry of replicating virus into Vero cells. Phenylpropanoids, flavonoids, and triterpenes were identified as the major phytochemical constituents in S. altissima and P. aquilinum.
Shin-Ya et al. (2023)	In vitro and in silico	Green tea, Matcha, black tea	Infectivity assay, antibody-based inhibitor screening kit, molecular docking	All three tea varieties effectively decreased the infectivity of Omicron subvariants BA.1, BA.2, XE, BA.5, BA.2.75, XBB.1, and BQ.1.1. Bioactive components of tea including the catechin EGCG and the theaflavin TFDG also decreased variant infectivity and blocked the interaction of BA.1 S RBD with ACE2.
Solo and Doss (2021)	In silico	North-East Indian medicinal plants	Molecular docking, molecular dynamics	50 phytochemicals from various plants displayed high binding affinity for Delta variant S. Of these, the top 15 compounds were flavones. 3,5,3′-Trimethoxy-6,7:4′,5′-bis(methylenedioxy)flavone from Nicotiana plumbaginifolia had the highest overall affinity for the S RBD (-8.7 kcal/mol).
Suručić et al. (2022)	In vitro and in silico	Alchemilla viridiflora	LC-MS, molecular docking, molecular dynamics, infectivity assay	Ellagitannins and flavonoids isolated from A. viridiflora extracts interacted favorably with S protein residues, with quercetin-3-(6”-ferulylglucoside) displaying the highest affinity (-8.035 kcal/mol). A. viridiflora methanol extract was also able to block SARS-CoV-2 entry in vitro (87.1%).
Wang et al. (2022)	In vitro and in silico	Peimine and other phytochemicals	Pseudovirus assay, fluorescence resonance energy transfer (FRET) assay, LC-MS, molecular docking	Peimine inhibited the entry of spike pseudoviruses into multiple cell lines by disrupting protein-protein interactions at the S-ACE2 interface. Activity extended to wild-type, B.1.1.7, and 501Y.V2 variants with EC50 values of 0.45 µM, 0.42 µM, and 0.43 µM respectively. Crude Fritillaria spp. extracts containing varying concentrations of peimine also inhibited entry.
Yi et al. (2022)	In vitro and in silico	Triterpenoids from Chinese liquorice (Glycyrrhiza uralensis)	Molecular docking, ELISA, pseudovirus assay, infectivity assay, immunofluorescence assay (IFA), SPR analysis	Glycyrrhetinic acid (GA) and licorice-saponin A3 (A3) displayed strong affinity for S RBD and inhibited the S-ACE2 interaction with respective IC50 values of 10.9 µM and 8.3 µM. GA and A3 also inhibited pseudovirus entry (EC50 = 4.98 µM, 9.30 µM) and live SARS-CoV-2 entry (EC50 = 3.17 µM, 75 nM).
Zhan et al. (2021)	In vitro and in silico	Flavonoids from sea buckthorn	Chromatography, SPR analysis, pseudovirus assay, molecular docking	Quercetin and isorhamnetin, major active flavonoids in sea buckthorn, displayed high affinity for ACE2. Isorhamnetin had the stronger affinity (KD = 2.51 ± 0.68 µM) and interacted with three key residues of ACE2, enabling it to block the entry of spike pseudovirus.
Zhang et al. (2021)	In vitro and in silico	Library of NP-derived small molecules	Molecular docking, BLI assay, antibody-based inhibitor screening kit, pseudovirus assay, infectivity assay	14 compounds interacted strongly with both S RBD and ACE2. Of these, eight compounds blocked the S-RBD interaction. EGCG, isobavachalcone (Ibvc) and salvianolic acid A (SalA) blocked infection of HEK293 cells by D614G, N501Y, N439K, and Y453F variant spike pseudoviruses and live SARS-CoV-2 by targeting three pockets on the S RBD and interacting with ACE2. The most potent inhibitor, EGCG, inhibited pseudovirus entry with an EC50 of 0.3209 µM and live SARS-CoV-2 entry with an EC50 of 9.415 µM.
Zhu et al. (2023)	In vitro and in silico	Luteolin	ELISA, pseudovirus assay, SPR analysis, ACE2 activity assay, molecular docking	Luteolin significantly inhibited the S-ACE2 interaction (IC50 = 0.61 mM) and reduced Delta and Omicron pseudovirus entry by disrupting interactions between key residues. The flavonoid bound to both S RBD and ACE2, with a slight preference for ACE2. Luteolin also significantly reduced the activity of ACE2.