In vitro and in silico Antiviral Activity of Biflavonoids From Garcinia madruno Against Mpox Virus

Abstract

Aim

Although current human transmission of monkeypox virus (MPXV) remains low, therapeutic options are limited. Biflavonoids, known for their antiviral properties, were evaluated in vitro and in silico against MPXV.

Procedures

Three biflavonoids; Morelloflavone (Mo), Volkensiflavone (Vo), and Fukugiside (Fu); isolated from Garcinia madruno, were tested at concentrations of 1.6, 3.1, 6.3, and 12.5 µM against an MPXV strain isolated in Medellín, Colombia (2022). Combined, pre-, co-, and post-infection treatment strategies were assessed in Vero-E6 cells at a multiplicity of infection (MOI) of 2. Cytotoxicity, plaque reduction assays, and molecular docking analyses were performed.

Results and Discussion

None of the biflavonoids exhibited cytotoxicity at concentrations up to 100 µM. All reduced MPXV infection by >70.9% across all concentrations under combined treatment strategies. In individual strategies at 6.3 µM, Mo, Vo, and Fu inhibited viral attachment by 59.4%, 79.3%, and 77.8%, respectively. Vo and Fu reduced the infection at co-treatment by 93.7% and 95.1%, respectively. Mo and Fu inhibited infection at post-entry steps by 64.4% and 56.7%, respectively. Molecular docking analyses revealed strong binding affinities (<–7.7 kcal/mol) between all biflavonoids and the MPXV proteins Methyltransferase and Poxin. Mo showed the most favorable binding energy (–8.4 kcal/mol), while Fu formed up to 12 hydrogen bonds with two catalytic residues of Methyltransferase. These findings indicate that biflavonoids could inhibit MPXV via distinct mechanisms: Mo affects both attachment and post-entry steps, while Vo and Fu show a possible direct activity against this virus and reduce attachment. Further studies are needed to validate their effects and clarify their therapeutic potential.

Keywords

MPXV biflavonoids antiviral molecular docking

1. Introduction

Mpox (formerly known as Monkeypox) is a zoonotic viral disease caused by the Mpox virus (MPXV), a member of the Poxviridae family, Orthopoxvirus genus.¹ MPXV is a double-stranded DNA virus (∼196,858 bp, ∼200 genes) with two major clades: Clade I, responsible for sustainable transmission from human to human in the Democratic Republic of Congo, and sporadic cases in several regions around the world. Clade II is endemic in West Africa and caused the 2022 outbreak.²

Although its life cycle is not fully understood, MPXV, like other poxviruses, is thought to enter host cells via macropinocytosis or direct membrane fusion, using glycosaminoglycans and laminin as attachment factors. Once inside, replication occurs entirely in the cytoplasm, relying on several viral enzymes and specialized replication factories.³

Since its reemergence in 2022, Mpox has remained a significant public health concern. As of March 21, 2026, the World Health Organization had reported more than 181,000 confirmed cases and 492 deaths worldwide. While most cases are self-limiting, immunocompromised individuals face a higher risk of severe complications.² Although therapeutic options remain somewhat limited, Tecovirimat has received formal approval for the treatment of orthopoxviruses in certain regions, such as the European Union, though it remains under investigational protocols elsewhere.⁴ Furthermore, while agents such as Brincidofovir have been used, concerns about the emergence of resistant strains and safety profiles continue to complicate the clinical landscape.⁵

Given this scenario, the search for novel antiviral candidates is critical. Natural products, including biflavonoids, have demonstrated a wide range of biological activities, such as antiproliferative, antioxidant, anti-inflammatory, cardioprotective, antiatherogenic, antibacterial, and antiviral effects.^6-12 Garcinia madruno, commonly known as madroño, ocoró, or canime, is a native tree from Latin America¹³ and belongs to the Clusiaceae family. Traditionally used in indigenous medicine, G. madruno has a complex matrix of bioactive compounds, among which biflavonoids are particularly prominent.^14,15 Among them, three compounds—Morelloflavone (Mo), Volkensiflavone (Vo), and Fukugiside (Fu)—have been previously isolated and characterized.^6,16 Mo, a flavanone-(3-8″)-flavone type biflavonoid (Figure 1A), has demonstrated anti-Human immunodeficiency virus (HIV) activity.⁸ Vo, with 3-8″ type interflavonoid linkage with two A₂B₂ systems (Figure 1B), has exhibited activity against influenza B virus.¹⁷ And Fu, a morelloflavone glucoside (Figure 1C), exhibits increased polarity due to its glycosidic structure and hydroxyl groups, although to the best of our knowledge, its antiviral potential remains unexplored.

Figure 1.

Chemical structure and cytotoxicity of biflavonoids isolated from G. madruno. Morelloflavone (A, D); Volkensiflavone (B, E); and Fukugiside (C, F). The viability of Vero-E6 in the presence of these compounds was determined by the MTT method (n=8). Concentrations from 3.1 to 100 µM were evaluated. Data was presented as Mean ± Standard Deviation (SD). The viability percentages of the treated cells were calculated based on a control of cells without treatment (100% of viability).

In addition to the properties already described, biflavonoids are dimeric flavonoids formed by the oxidative coupling of two monoflavonoid units. Because they are essentially double molecules, their physicochemical properties are distinct and often push the boundaries of traditional drug-likeness rules, such as Lipinski’s Rule of Five, which frequently do not apply to complex natural products.¹⁸ Furthermore, their dimeric architecture can confer greater biological potency than their monomeric counterparts.¹⁹ Therefore, in vitro experimental evaluation is essential to assess the bioactive potential of these compounds accurately.²⁰

Other biflavonoids have demonstrated antiviral activity in vitro against RNA viruses as Hepatitis C virus (HCV),²¹ severe acute respiratory syndrome coronavirus (SARS-CoV) ²², Influenza A virus (IAV),²³ Dengue virus (DENV),²⁴ HIV⁹ and DNA viruses as Herpes virus (HSV), varicella-zoster virus (VZV).¹⁸ Despite growing evidence supporting the antiviral potential of biflavonoids, data on their activity against MPXV remain limited. Therefore, we aimed to investigate the in vitro effects of these three biflavonoids Mo, Fu, and Vo, against MPXV and to explore their potential molecular interactions with three viral proteins through docking analyses.

2. Materials and Methods

2.1. In Vitro Evaluations

2.1.1. Cell Culture, Virus, and Compounds

Vero E6 cells (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA), supplemented with 2% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) and 1% penicillin/streptomycin (pen/strep; Sigma, St. Louis, MO, USA). Cells were maintained at 37°C in a humidified incubator with 5% CO₂ and were used for cytotoxicity assays, antiviral evaluations, and virus propagation.

Viral infections were performed using a 2022 Colombian MPXV isolate (grupo Inmunovirología, Universidad de Antioquia, Medellín-Colombia), Colombia); the complete genome sequence is available in the GISAID database (EPI_ISL_20219588), and the formal characterization of this isolate was recently published ²⁵. The virus was propagated on Vero E6 cells and incubated for 4 days at 37 °C with 5% CO₂. Viral titers were determined by plaque assay.

Three biflavonoids isolated from G. madruno (Mo, Fu, and Vo) were previously characterized.^16,17 The lyophilized compounds were dissolved in Phosphate Buffered Saline (PBS, Lonza, Rockland, ME, USA) containing 10% dimethyl sulfoxide (DMSO; Supelco, Darmstadt, Germany), prepared to ensure a maximum working DMSO concentration <0.6%, minimizing any potential cytotoxic or off-target effects on the cells. The compounds were used at concentrations between 1.6 and 100 µM, according to the test.

2.1.2. Cytotoxicity Assay

Vero E6 cell viability was assessed by MTT assay (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl diphenyltetrazolium bromide; Sigma. St Louis, USA), following the manufacturer’s instructions. Briefly, monolayers of 3×10⁴ cells/well cells seeded in 96-well plates were treated with biflavonoids (3.1 to 100 µM) for 48 h at 37°C with 5% CO₂. MTT at 2 mg/mL was added for 2 h at 37 °C, and formazan crystals were solubilized in DMSO. Absorbance was measured at 570 nm. Viability was expressed as a percentage relative to untreated controls. Data are presented as mean ± standard deviation of two independent experiments in quadruplicate (n=8).

2.1.3. Antiviral Assay

Antiviral activity was evaluated in Vero E6 monolayers using four strategies described below. Supernatants from all assays, except for the attachment assay, were stored at -80°C until viral titration by plaque assay. Infected but untreated cells served as virus control (VC). Two independent experiments in quadruplicate were conducted (n=8). Each experimental unit was performed independently on different days, by different operators, and using independent cell cultures to ensure the reproducibility of the assays.

2.1.3.1 Combined Strategy

Four non-cytotoxic concentrations (1.6, 3.1, 6.3, and 12.5 µM) of the compounds were selected based on previous reports showing antiviral activity of flavonoids at low micromolar concentrations.¹³ Monolayers in 96-well plates were pre-treated with Mo, Vo, or Fu for 1h at 37°C. After removing the supernatants, cells were infected with MPXV at a multiplicity of infection (MOI) of 2 in the presence of the same biflavonoid concentrations and incubated for 1h at 37°C. The viral inoculum was removed, fresh medium containing the corresponding biflavonoids was added, and cells were incubated for 48 h. Rifampicin (50 µM) was used as a positive control of viral inhibition.²⁶

2.1.3.2. Attachment Strategy

Monolayers in 48-well plates were pre-cooled at 4°C for 30 min. Subsequently, biflavonoids (6.3 µM) and MPXV (60 PFU/well) were mixed 1:1 and added to the cells, followed by a 1h incubation at 4 °C. After washing with cold PBS, cells were incubated with semi-solid overlay (2% carboxymethyl-cellulose in DMEM with 2% FBS and 1% pen/strep) at 37°C until plaque formation. Finally, the cells were washed, fixed, stained, and analyzed as described in section 2.1.4. Heparin (100 μg/mL) was used as a positive control of the attachment block.²⁷

2.1.3.3. Co-Treatment Strategy

Each biflavonoid (6.3 µM) was mixed 1:1 with MPXV (MOI: 2) and incubated for 1h at 4°C. Subsequently, the mixtures were added to monolayers in 96-well plates and incubated for 1 h at 37°C. After inoculum removal, fresh medium was added, and cells were incubated for 48h. Heparin (100 μg/mL) served as a positive control of viral inhibition.²⁷

2.1.3.4. Post-Treatment

Monolayers in 96-well plates were infected with MPXV (MOI: 2) for 1h. After removing the viral inoculum, medium containing each biflavonoid (6.3 µM) was added, and cells were incubated for 48 h. Rifampicin (50 µM) was used as a positive control of viral inhibition.²⁶

2.1.4. Quantification of Infectious Viral Particles by Plaque Assay

Viral titers were determined by plaque assay. Briefly, Vero E6 monolayers (1.5x10⁵ cells/well) in 24-well plates were inoculated with serial dilutions of the supernatants. After 1 h of incubation, the inoculum was removed and replaced with 1 mL of semi-solid overlay. After 3 days at 37°C, cells were fixed and stained with 4% formaldehyde/1% crystal violet, and plaques were counted. Viral titers were expressed as plaque-forming units per milliliter (PFU/mL), and inhibition percentages were calculated relative to the untreated virus controls.

2.1.5. Statistical Analysis

Data normality was determined using the Shapiro-Wilk test. Differences in viral titers between treatment and VC were analyzed using a Student’s t-test (for normally distributed data) or a Mann-Whitney U test (for non-normally distributed data). One-way ANOVA or Kruskal–Wallis test was used to analyze two or more concentrations for each compound. Statistical analyses were performed with the GraphPad Prism® 8.01 package for Windows™ (GraphPad Software, San Diego, CA, USA). P-values lower than 0.05 were considered statistically significant.

2.2. In Silico Evaluations

2.2.1. Target Protein Selection and Preparation

The selection of viral targets was based on their essential and non-redundant roles at different stages of the MPXV life cycle, aiming to correlate the computational findings with the observed in vitro inhibition patterns. Specifically, we selected the Methyltransferase (PDB: 8CEV) for its role in mRNA capping;²⁸ the Helicase-Primase D5 (PDB: 8HWG) as a key enzyme for genome replication;²⁹ and Poxin (PDB: 8ORV) for its function in evading the host’s innate immune response.³⁰ Protein preparation was conducted using the AutoDockTools (ADT) suite. The process involved removing crystallographic water molecules, adding missing hydrogen atoms, and assigning Kollman united-atom partial charges to the macromolecule to achieve a more accurate representation of the protein’s electrostatic potential. Finally, non-polar hydrogen atoms were merged, and the structures were exported in PDBQT format for use in AutoDock Vina software version 1.1.2 (Scripps Research Institute, San Diego, CA, USA).³¹

2.2.2. Ligand Selection

The 3D structures of Mo (CID: 5319895), Vo (CID: 23844069), and Fu (CID: 11968471) were retrieved from the PubChem database. To ensure structural stability, the geometry of each ligand was optimized through energy minimization using the Universal Force Field (UFF). Following minimization, ligand flexibility and active torsions were assigned using the Python Molecular Viewer (PMV), a component of the ADT package. The structures were then converted to PDBQT format, incorporating Gasteiger partial charges and polar hydrogen atoms, for subsequent docking simulations with AutoDock Vina.

2.2.3. Molecular Docking

Target protein-ligand interactions were analyzed in triplicate through independent docking runs using AutoDock Vina (version 1.1.2). To identify target hotspots, we cross-referenced the binding and active site residues reported in the Protein Data Bank (PDB) with pocket predictions from two online tools: PeptiMap (Boston University, USA)³² and ProteinsPlus (Universität Hamburg, Germany).³³ This integrated approach, combining experimental data with dual computational predictions, ensured that the selected docking sites were accurately validated (Supplementary Table 1). Based on the predicted sites, grid box dimensions X-Y-Z were defined as 30 Å, with an exhaustiveness value of 10 (coordinates for each protein are provided in Supplementary Table 1). The docking protocol was validated by redocking the co-crystallized ligands and including reference antiviral compounds as positive controls for each target: Tecovirimat for Methyltransferase,³⁴ Caffeic acid for Poxin,³⁵ and Rifampicin for Helicase-primase D5²⁴. These controls allowed for a direct comparison and assessment of the significance of the binding scores obtained for the biflavonoids (Figure 5). Hydrogen bonds and hydrophobic interactions were further examined using two-dimensional interaction diagrams generated with LigPlot v2.2 (EMBL-EBI, Wellcome Genome Campus, Hinxton.³⁶ The 3D interactions were visualized using PyMoL version 3.1 (San Carlos, California, USA).

2.2.4. ADMET Profiling

To provide a preliminary assessment of the pharmacokinetic and toxicological profiles of the studied biflavonoids, in silico screening was performed using the SwissADME web server (Swiss Institute of Bioinformatics, Switzerland). Molecular structures were converted into Canonical SMILES format to evaluate potential medicinal chemistry alerts.³⁷ The complete ADMET reports for each compound are provided in the supplementary Figure 3.

3. Results

3.1. Biflavonoids Derived From Garcinia madruno are Not Cytotoxic

The cytotoxicity of Mo, Vo, and Fu was determined using the MTT assay. All biflavonoids were non-cytotoxic at the highest concentration evaluated of 100 µM, after 48 h of treatment (Figure 1D–F). No dose-dependent effect of viability was observed, and this pattern was consistent across the three biflavonoids, despite their structural differences (Figure 1A–C). Due to the absence of cytotoxicity at tested concentrations, the biflavonoids were further evaluated for their antiviral potential.

3.2. Biflavonoids Inhibits MPXV Infectious Particles Production at Various Concentrations Through a Combined Antiviral Strategy

The biflavonoids were evaluated against MPXV using a combined treatment strategy involving administration before, during, and after infection, and viral titers were compared to the viral control (VC). Mo, Vo, and Fu significantly reduced MPXV infection by 71–85.1%, 85–93.7%, and 81.6–81.3%, respectively, at concentrations ranging from 1.6 to 12.5 µM (Figure 2). An inhibition percentage of 68% was obtained after treatment with the positive control of viral inhibition, Rifampicin. A working concentration of 6.3 µM for each biflavonoid was selected for further experiments, as the antiviral effects were comparable across the tested concentrations, and compound availability was limited.

Figure 2.

Anti-MPXV activity in the combined strategy. Vero-E6 cells were treated with three compounds: Mo (A), Vo (B), and Fu (C), four concentrations of each one (1.6-12.5 µM), before, during, and after the infection with MPXV. Rifampicin (50 µM) was used as a positive control of viral inhibition. The asterisks indicate statistically significant differences with VC (****p< 0.0001; ANOVA), and error bars indicate standard error of the mean (n=8).

3.3. Inhibition of Infectious MPXV Particles by Biflavonoids Depends on the Individual Strategy

Viral attachment was significantly inhibited by Mo, Vo, and Fu at 6.3 µM, with reductions of 59.4%, 79.3%, and 77.8%, respectively. Heparin (positive control for viral inhibition) showed a 84.1% reduction (Figure 3A). In the co-treatment assay, Vo and Fu reduced infectious MPXV particle production by 93.7% and 95.1%, respectively. Mo showed no significant effect. The positive control (Heparin) reduced the MPXV infection by 82.3% using this antiviral strategy (Figure 3B). In post-entry assays, Mo and Fu reduced infection by 64.4% and 56.7%, respectively, while Vo had no effect. Rifampicin (positive control of viral inhibition) reduced viral infectivity by 81.4% (Figure 3C).

Figure 3.

Antiviral effects by individual strategy. Vero-E6 cells were infected with MPXV and treated with Mo, Vo, and Fu (6.3 µM) through three individual strategies: Attachment strategy (A), co-treatment strategy (B), and post-treatment strategy (C). Heparin and Rifampicin were used as positive controls of viral inhibition. The asterisks indicate statistically significant differences compared to the viral control -VC (**** p < 0.0001; Mann-Whitney), and error bars indicate standard error of the mean; n=8.

3.4. The Binding Energies of Three Biflavonoids and Two Proteins of MPXV Are Favorable According to Molecular Docking

The prediction of the interaction between the Melthyltrasferasa protein (PDB ID: 8CEV) and the biflavonoids (Mo-Vo-Fu) resulted in favorable binding free energies. Mo binding energy was −8.4 kcal/mol and presented three hydrogen bonds, two with the same amino acid (Asp95) and another with Gly72. Vo was -8.1 Kcal/mol with two hydrogen bonds (Asp95 and Gly72); and Fu was -8.1 Kcal/mol with 12 hydrogen bonds (Lys41, Gly68, Ala70, Asp95, Arg97 (x3), Asp138 (x2), Arg140, Ser141, and Arg177). Likewise, the Poxin protein binding energies ranged from −7.7 to −8.3 Kcal/mol. Mo formed four hydrogen bonds (Thr93, Arg95 (x2), Ile193), Vo formed three (Arg95 (x2), Ile193), and Fu formed four (Thr93 (x3), Ile193). On the other hand, helicase-primase D5 (PDB ID: 8HW6) showed higher binding energies with Mo and Vo (−8.3 and −8.0 Kcal/mol) in comparison with Fu (−6.1 Kcal/mol). All three isolated biflavonoids exhibited hydrophobic interactions with MPXV´s proteins (Table 1-Figure 4). The docking protocol was validated by redocking the co-crystallized ligand, which successfully recapitulated the experimental binding pose (Figure 5).

Table 1.

Molecular Docking and in silico Interactions Between MPXV and Each Compound

Ligand	Protein	Free binding energy (Kcal/mol)	Hydrogen bonds	Residues forming H bonds	Distance between H+ bonds (Å)	Residues participating in hydrophobic interactions
Morelloflavone	Methyltransferase VP39 PDB: 8CEV	−8.4 ± 0.00	3	Gly72	3,06	Gly38-Gln39-Leu42-Gly68-Ser69-Ala70-Pro71-His74-Arg97-Phe115-Asp138-Arg140-Ser141
	Methyltransferase VP39 PDB: 8CEV	−8.4 ± 0.00	3	Asp95 (x2)	2,80-2,85
	Poxin PDB: 8ORV	−8.3 ± 0.00	4	Thr93	3,01	Ala8-Asn54-Ile77-Tyr94-Leu191-Pro192-Pro194
				Arg95 (x2)	2,89-3,14
				Ile193	2,98
	helicase-primase D5 PDB: 8HWG	−8.3 ± 0.2	5	Lys509	2.95	Ala506-Gly508-Thr511-His629-Ser631-Leu651-Leu655-Asp-656
				Ser510	3.11
				Phe630	2.90
				Gln632 (x2)	3.04-3.35
Volkensiflavone	Methyltransferase VP39 PDB: 8CEV	−8.1 ± 0.00	2	Gly72	3,06	Gly38-Gln39-Leu42-Gly68-Ser69-Ala70-Pro71-His74-Arg97-Phe115-Asp138-Arg140-Ser141
	Methyltransferase VP39 PDB: 8CEV	−8.1 ± 0.00	2	Asp95	2,71
	Poxin PDB: 8ORV	−8.0 ± 0.00	3	Arg95 (x2)	2,91-3,12	Ala8-Asn54-Thr93Tyr94-His121-Leu191-Pro192-Pro194
	Poxin PDB: 8ORV	−8.0 ± 0.00	3	Ile193	2,94	Ala8-Asn54-Thr93Tyr94-His121-Leu191-Pro192-Pro194
	helicase-primase D5 PDB: 8HWG	−8.0 ± 0.2	3	Lys509	2.89	Ala506-Gly508-Thr511-His629-Ser631-Gln632-Leu651-Leu655
				Ser510	3.06
				Phe630	2.89
Fukugiside	Methyltransferase VP39 PDB: 8CEV	−8.1 ± 0.00	12	Lys41	2,8	Glu147-Pro148-Lys175
				Gly68	3,04
				Ala70	2,74
				Asp95	3,13
				Arg97 (x3)	3,34-3,22-3,81
				Asp138 (x2)	2,93-3,03
				Arg140	3,26
				Ser141	3,02
				Arg177	3,10
	Poxin PDB: 8ORV	−7.7 ± 0.23	4	Thr93 (x3)	3,28-3,14-3,15	His7-Ala8-Asn54-Ser55-Gln56-Ile77-Thr93-Tyr94-Pro192-Pro194
	Poxin PDB: 8ORV	−7.7 ± 0.23	4	Ile193	2,94	His7-Ala8-Asn54-Ser55-Gln56-Ile77-Thr93-Tyr94-Pro192-Pro194
	helicase-primase D5 PDB: 8HWG	−6.1 ± 0.1	2	Asn463	3.18	Glu459-Glu460-Asp467-Phe630-Lys649-Asp652-Leu655-Lys658
	helicase-primase D5 PDB: 8HWG	−6.1 ± 0.1	2	Ile464	3.29	Glu459-Glu460-Asp467-Phe630-Lys649-Asp652-Leu655-Lys658

Figure 4.

Molecular docking between compounds and three MPXV proteins. The interactions formed between Methyltransferase and Mo (A), Vo (B), and Fu (C); between Poxin and Mo (D), Vo (E), and Fu (F); also, between helicase-primase D5 and Mo (G), Vo (H), and Fu (I)are included. The 2D figures schematize hydrophobic contacts as multiple red fanning lines, and hydrogen bonds and their length as green dotted lines. The 3D and 2D graphics were obtained by PyMoL and LigPlot®, respectively.

Control ligands previously reported (rifampicin,²⁶ tecovirimat,³⁴ and caffeic acid³⁵ showed favorable binding energies (-7.7, -7.6, and -5.9 kcal/mol) at the target sites of VP39, Helicase-primase D5, and Poxin, respectively. Additionally, redocking the co-crystallized ligands successfully reproduced the experimental binding modes with favorable energy scores, further validating the accuracy of the in silico protocol (Figure 5).

Figure 5.

Molecular docking validation and comparative binding analysis. Panel A: Comparative positioning of Methyltranferase and tecovirimat/inhibitor TO1119; Poxin and caffeic acid/agonist MD1203; Helicaseprimase D5 and rifampicin/ssDNA. The inhibin controls previously reported in the literature are shown in red, and the co-crystallized ligand of each protein is shown in blue. Panel B: Redocking validation. The native co-crystallized pose is represented by the blue dotted mesh, while the redocked pose obtained in this study is shown in red. The low RMSD values (all < 2.0 Å) confirmed the reliability of the docking parameters and the scoring function’s ability to replicate experimental binding modes.

4. Discussion

MPXV caused a global outbreak between 2022 and 2023 and continues to circulate sporadically in several regions, driven by international travel, sexual transmission, and close contact. Although antivirals like Tecovirimat and Brincidofovir are available for severe cases, their limitations—including resistance, toxicity, and restricted access—highlight the need for new therapeutic options. Natural compounds like biflavonoids from tropical plants like Garcinia madruno, with reported anti-inflammatory, antiviral, and immunomodulatory properties^16,38-40 represent potential candidates against this virus.

In this study, three biflavonoids (Mo, Vo, and Fu) isolated from G. madruno were evaluated against a Colombian MPXV isolate in Vero E6 cells. All three compounds exhibited antiviral activity at the tested concentrations without inducing cytotoxicity, maintaining nearly 100% cell viability at concentrations up to 100 µM. This favorable safety profile is consistent with previous in vitro and in vivo reports for this class of bioflavonoids at comparable concentrations,^16,17 and it may be linked to their antioxidant properties, such as free radical neutralization^41,42 and the modulation of the Nrf2-mediated antioxidant response.^43,44

However, these experimental observations contrast with the in silico ADMET profiling, which predicted potential toxicity and a low bioavailability score (<0.55)⁴⁵ (Figure supplementary 3). This discrepancy highlights that ADMET predictors are probabilistic models, not biological certainties; such algorithms often overestimate toxicity alerts by identifying reactive hydroxyl groups or planar aromatic rings, common in flavonoids, without considering the complex metabolic detoxification.⁴⁶ In living organisms, these molecules undergo efficient Phase II metabolism (like glucuronidation or sulfation), which mitigates the risks predicted by ADMET and aligns with the lack of toxicity observed in our assays and previous murine studies with similar molecules.^17,47,48

Remarkably, the three biflavonoids inhibited MPXV infection by over 90% at all tested concentrations following combined treatment. These inhibition levels were notably higher than those achieved with individual treatment strategies (Figure 2). The antiviral effect was sustained even at a high MOI of 2, which was selected to ensure a sufficient recovery of viral titers by plaque assay after antiviral assays. Although high MOIs can sometimes mask antiviral activity by infecting nearly all cells simultaneously and reducing assay sensitivity, the compounds in this case maintained their inhibitory effect even under such challenging conditions.⁴⁹

Our findings suggest a synergistic effect of the compounds when present at different stages of the viral replicative cycle. Interestingly, Vo had previously shown antiviral activity only against the influenza B virus, while being inactive against other viruses tested. Indeed, other biflavonoids exhibited antiviral activity depending on the viral model.^18,50

It is worth noting that for Fu, a dose-independent response was observed (Figure 2). This result suggests that once the compound reaches a threshold sufficient to block the replicative cycle, the reduction in viral activity remains virtually unchanged at higher concentrations within the tested range.⁵¹

The antiviral activity observed for all three biflavonoids (Figure 2) may be attributed to their shared flavone-based core structure, which likely functions as a pharmacophore driving this effect, as reported in models of metabolic and systemic diseases.^52,53 Additionally, their antiviral activity may involve modulation of lipid metabolism, potentially altering membrane dynamics and affecting viral entry, budding, or envelope formation—key processes in the MPXV life cycle, particularly for the extracellular enveloped virion (EEV), which carries an additional outer lipid envelope, unlike intracellular mature virions (IMV) that possess only a single envelope.^54-56

The enhanced antiviral activity observed with the combined strategy may be partly explained by interference with viral attachment by all biflavonoids, as observed in the viral binding assays (Figure 3A). This effect could result from interactions with cellular receptors or viral glycoproteins.^57,58 Glycosaminoglycans have been proposed as primary attachment factors for MPXV, and heparin—used as a positive control in our assays—is known to bind cellular glycosaminoglycans. In our system, heparin effectively reduced infection by interfering with viral attachment, suggesting that biflavonoids may act through a similar mechanism.^59,60 Other biflavonoids have also demonstrated interference with viral binding, such as those targeting Influenza A entry proteins.²³

We also identified, in co-treatment assays, a possible direct effect of Vo and Fu against MPXV (Figure 3B), a mechanism reported for another biflavonoid, tetrahydroamentoflavone.⁶¹ Flavonoids, in general, have been more extensively studied and shown broad virucidal activity against viruses such as DENV-2,⁶² ZIKV,⁶³ CHIKV,⁶⁴ and SARS-CoV-2.²² This effect could be due to interactions with components of the viral envelope, including viral glycoproteins, altering its structure and stability, preventing the virus from binding to host cells, releasing its genetic material into the cell, or decreasing the replicative capacity of the viral particle. In contrast, the results of co-treatment with Mo suggest that the inhibition is more closely related to interactions with the cellular receptor than to the viral particle itself (Figure 3B). Future assays that include the evaluation of the affinity for cellular receptors and viral glycoproteins could confirm this hypothesis.

On the other hand, Mo and Fu inhibited the post-entry stages of MPXV infection (Figure 3C), suggesting that the affected steps in the viral cycle may include viral genome replication, assembly, maturation, and/or release of infectious viral particles. These observations align with previous reports on flavonoids, such as Apigenin, which inhibits buffalopox virus (BPXV) by affecting viral genome replication and protein synthesis.⁶⁵ Similarly, myricetin and (–)-gallocatechin have been identified as potent inhibitors of the MPXV H1 phosphatase, an essential enzyme for viral immune evasion.⁶⁶

To explore the molecular basis of this post-entry inhibition, we conducted molecular docking against three key viral targets: Methyltransferase, Poxin, and Helicase-primase (D5). These proteins were selected due to their essential roles in the MPXV life cycle: Methyltransferase is crucial for mRNA capping and protein modification²⁸; Poxin acts as a nuclease that degrades cGAMP to evade the host’s interferon response³⁵; and the D5 Helicase-primase (also known as E5 or OPG117) is fundamental for DNA synthesis in the host cytoplasm.^29,30 To ensure the biological relevance of these in silico models, we applied a strict binding affinity threshold of ≤ -7.0 kcal/mol, a value associated with high-affinity interactions in AutoDock Vina.⁶⁷ The docking protocol was validated by redocking the co-crystallized ligand, effectively reproducing the experimental binding pose (Figure 5).

Our results indicate that the analyzed biflavonoids interact favorably with the target proteins, exceeding the established reference benchmarks. These findings provide a computational rationale and a putative starting point to explain the observed in vitro activity through potential enzymatic inhibition. Notably, Mo exhibited the highest overall affinity, with binding energies reaching <-8.3 Kcal/mol for both Methyltransferase and Poxin (Table 1, Figure 4). Regarding the Methyltransferase, these biflavonoids likely interfere with the enzyme’s catalytic site, potentially compromising the post-translational modifications of viral proteins and glycolipids, thereby impairing viral assembly and maturation.²⁸ Specifically, Asp95, a residue critical for binding the ribose ring within the active site, was predicted to form hydrogen bonds with all three biflavonoids. Fu formed an additional eleven hydrogen bonds, including with the key catalytic residue Lys41, which may further disrupt enzyme function and impair genome replication.

Regarding the Helicase-primase D5, both Mo and Vo exhibit favorable binding energies by interacting with amino acids within known catalytic motifs.²⁹ Since D5 is essential for unwinding double-stranded DNA and synthesizing RNA primers, its potential inhibition could arrest viral replication. However, the distinct binding orientations observed for Mo and Vo suggest divergent putative inhibitory mechanisms (Supplementary Figure 2), hypotheses that require further biochemical validation.

Mo and Vo, due to their smaller chemical structure, are located directly in a more internal binding pocket of the protein D5. This deep spatial coupling within the catalytic site prevents the natural substrate from binding, suggesting that Mo and Vo may act as competitive inhibitors. While Fu, with its carbohydrate, is situated in a more external binding area of the protein. This position suggests a potential dual mechanism of inhibition. Its external binding could induce a conformational change that disrupts the active site (allosteric inhibition). Simultaneously, its interactions with specific amino acids within the catalytic motif indicate it can also directly influence the catalytic function, suggesting a secondary, competitive-like effect.

On the other hand, poxin is a viral nuclease that degrades double-stranded RNA, thereby evading intracellular pattern recognition receptors (PRRs), such as the cGAS-STING pathway, and reducing the immune response. In our results, stable interactions with Mo and Fu were found. These interactions were favored by hydrogen bonds formed with Arg95 and Ile193 (Table 1, Figure 4), which, although located outside the catalytic site, may induce allosteric interference that could prevent the enzyme from functioning correctly.

In the molecular docking analysis of the three viral proteins, both the positive controls and co-crystallized ligands exhibited high binding affinities (Figure 5). These interactions were modeled using optimized coordinates that were validated by cross-referencing PDB experimental data with predictions from PeptiMap and ProteinsPlus (Supplementary Table 1). The observation that binding energies for these controls were comparable to those of the test molecules (Table 1) confirms the reliability of our docking parameters and underscores the predictive accuracy of the model.

Some limitations of this study include the limited availability of biflavonoids, which prevented testing over a wider concentration range. Consequently, CC50, IC50, and Selectivity Index (SI) values could not be determined. Furthermore, while molecular docking provided valuable insights, more rigorous in silico analyses, such as molecular dynamics simulations, were beyond the current scope of this study due to limited access to the high-performance computing infrastructure required for long-atomistic simulations. Consequently, our findings rely on optimized docking protocols to identify the most favorable binding orientations as a basis for future mechanistic studies.

In summary, all three biflavonoids demonstrated antiviral activity against MPXV with minimal cytotoxicity at the tested concentrations, supporting further studies to evaluate their potential as candidates for MPXV therapy. While our in silico models suggest strong binding affinities for non-structural viral proteins (particularly Methyltransferase and Poxin), these findings serve as docking-based hypotheses regarding their potential targets. Specifically, the predicted interactions of Vo and Fu support the possibility of direct viral inhibition, which warrants further investigation into their use as topical treatments for MPXV-associated lesions. Further research is warranted to elucidate their precise mechanisms of action, in vivo assess efficacy, and explore their immunomodulatory potential, as previously reported.¹⁷

Supplemental Material

Supplemental Material - In Vitro and in Silico Antiviral Activity of Biflavonoids From Garcinia madruno Against Mpox Virus

Supplemental Material for In Vitro and in Silico Antiviral Activity of Biflavonoids From Garcinia madruno Against Mpox Virus by Laura M. Monsalve-Escudero, María I. Zapata-Cardona, Jorge H. Tabares-Guevara, Edison Osorio, Wildeman Zapata-Builes, Ana L. Rodriguez-Perea, Wbeimar Aguilar-Jimenez in Natural Product Communications.

Footnotes

Acknowledgements

Authors thank the Universidad de Antioquia for their support (sustainability program).

ORCID iDs

Laura M. Monsalve-Escudero

Wbeimar Aguilar-Jimenez

Consent to Participate

There are no human subjects in this article and informed consent is not applicable.

Author Contributions

Conceptualization: LMME, MIZC, JHTG, WAJ. Isolation and characterization of the compounds: EO. Experimental procedures: MIZC, LMME. Manuscript writing: LMME, MIZC, JHTG, WAJ, ALRP, WZB. All authors reviewed and accepted the final version of the manuscript.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Grant No. 2023-66230, jointly funded by Universidad Cooperativa de Colombia (UCC), Universidad de Antioquia (UdeA), and Corporación Universitaria Remington. The funding sources had no role in study design, data collection, analysis, interpretation, or manuscript preparation.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Supplemental Material

Supplemental Material for this article is available online.

References

Moghib

Asim

Salomon

Ghanm

Shivashankar

. Monkeypox: an in-depth examination of epidemiology, pathophysiology, and global public health implications. Ann Med Surg. 2025;87(4):2093-2104. doi:10.1097/MS9.0000000000003064.

CDC . Monkeypox in the United States and Around the World: Current Situation. Monkeypox; 2026. https://www.cdc.gov/monkeypox/situation-summary/index.html. Accessed March 23 2026.

Greseth

Traktman

. The Life Cycle of the Vaccinia Virus Genome. Annu Rev Virol. 2022;9(1):239-259. doi:10.1146/annurev-virology-091919-104752.

Organization

. Global Mpox Trends; 2026. https://worldhealthorg.shinyapps.io/mpx_global/. Accessed March 23, 2026.

Tecovirimat SIGA. European Medicines Agency (EMA). 2021. Accessed March 23, 2026.https://www.ema.europa.eu/en/medicines/human/EPAR/tecovirimat-siga

Viral

. Bacterial, and Fungal Infections - Chapter 59: Role of natural products in infectious diseases. Sustainable Development Goals - Resource Centre. 2024. https://sdgresources.relx.com/book-chapters/viral-parasitic-bacterial-and-fungal-infections-chapter-59-role-natural-products. Accessed March 23, 2026.

Commissioner O of the . Mpox. FDA. Published online March 26, 2025. https://www.fda.gov/emergency-preparedness-and-response/mcm-issues/mpox. Accessed March 23, 2026.

Ciumărnean

Milaciu

Runcan

, et al. The Effects of Flavonoids in Cardiovascular Diseases. Molecules. 2020;25(18):4320. doi:10.3390/molecules25184320.

Lin

Anderson

Flavin

, et al. In vitro anti-HIV activity of biflavonoids isolated from Rhus succedanea and Garcinia multiflora. J Nat Prod. 1997;60(9):884-888. doi:10.1021/np9700275.

10.

Manthey

. Biological properties of flavonoids pertaining to inflammation. Microcirculation. 2000;7(6 Pt 2):S29-S34.

11.

Šamec

Karalija

Dahija

Hassan

STS

. Biflavonoids: Important Contributions to the Health Benefits of Ginkgo (Ginkgo biloba L.). Plants. 2022;11(10):1381. doi:10.3390/plants11101381.

12.

Shamsudin

Ahmed

Mahmood

, et al. Antibacterial Effects of Flavonoids and Their Structure-Activity Relationship Study: A Comparative Interpretation. Molecules. 2022;27(4):1149. doi:10.3390/molecules27041149.

13.

Bastida

. PHYTOCHEMICAL CHARACTERIZATION OF A BIFLAVONOID FRACTION FROM Garcinia madruno: INHIBITION OF HUMAN LDL OXIDATION AND ITS FREE RADICAL SCAVENGING MECHANISM. Vitae. 2009;16(3):369-377. doi:10.17533/udea.vitae.3016.

14.

Ramirez

Gil

Marín-Loaiza

Rojano

Durango

. Chemical constituents and antioxidant activity of Garcinia madruno (Kunth) Hammel. J King Saud Univ – Sci. 2019;31(4):1283-1289. doi:10.1016/j.jksus.2018.07.017.

15.

Carrillo-Hormaza

Ramírez

Quintero-Ortiz

, et al. Comprehensive characterization and antioxidant activities of the main biflavonoids of Garcinia madruno: A novel tropical species for developing functional products. J Funct Foods. 2016;27:503-516. doi:10.1016/j.jff.2016.10.001.

16.

Sabogal-Guáqueta

Carrillo-Hormaza

Osorio

Cardona-Gómez

. Effects of biflavonoids from Garcinia madruno on a triple transgenic mouse model of Alzheimer’s disease. Pharmacol Res. 2018;129:128-138. doi:10.1016/j.phrs.2017.12.002.

17.

Tabares-Guevara

Lara-Guzmán

Londoño-Londoño

, et al. Natural Biflavonoids Modulate Macrophage-Oxidized LDL Interaction In Vitro and Promote Atheroprotection In Vivo. Front Immunol. 2017;8:923. doi:10.3389/fimmu.2017.00923.

18.

Lin

Flavin

Schure

, et al. Antiviral activities of biflavonoids. Planta Med. 1999;65(2):120-125. doi:10.1055/s-1999-13971.

19.

Thapa

Woo

Chi

, et al. Biflavonoids are superior to monoflavonoids in inhibiting amyloid-β toxicity and fibrillogenesis via accumulation of nontoxic oligomer-like structures. Biochemistry. 2011;50(13):2445-2455. doi:10.1021/bi101731d.

20.

Newman

Cragg

. Natural Products as Sources of New Drugs over the Nearly Four Decades from 01/1981 to 09/2019. J Nat Prod. 2020;83(3):770-803. doi:10.1021/acs.jnatprod.9b01285.

21.

Khachatoorian

Arumugaswami

Raychaudhuri

, et al. Divergent antiviral effects of bioflavonoids on the hepatitis C virus life cycle. Virology. 2012;433(2):346-355. doi:10.1016/j.virol.2012.08.029.

22.

Takeda

Tamura

Jamsransuren

Matsuda

Ogawa

. Severe Acute Respiratory Syndrome Coronavirus-2 Inactivation Activity of the Polyphenol-Rich Tea Leaf Extract with Concentrated Theaflavins and Other Virucidal Catechins. Molecules. 2021;26(16):4803. doi:10.3390/molecules26164803.

23.

Cho

Choi

Ahmad

Choi

. Antiviral Effect of Amentoflavone Against Influenza Viruses. Int J Mol Sci. 2024;25(22):12426. doi:10.3390/ijms252212426.

24.

Coulerie

Nour

Maciuk

, et al. Structure-activity relationship study of biflavonoids on the Dengue virus polymerase DENV-NS5 RdRp. Planta Med. 2013;79(14):1313-1318. doi:10.1055/s-0033-1350672.

25.

Zapata MI, Monsalve LM, Rodríguez AL, et al. Characterization of a Colombian Mpox virus isolate and exploratory evaluation of repurposed antiviral drugs. Biomédica. 2026;46 (4). 10.7705/biomedica.8247. Epub 2026 May 5, available on: https://revistabiomedica.org/index.php/biomedica/article/view/8247/5973.

26.

Structural basis for the inhibition of poxvirus assembly by the antibiotic rifampicin. PNAS. Accessed March 23, 2026. doi:

10.1073/pnas.1810398115

27.

Khanna

Ranasinghe

Jackson

Parish

. Heparan sulfate as a receptor for poxvirus infections and as a target for antiviral agents. J Gen Virol. 2017;98(10):2556-2568. doi:10.1099/jgv.0.000921.

28.

Silhan

Klima

Otava

, et al. Discovery and structural characterization of monkeypox virus methyltransferase VP39 inhibitors reveal similarities to SARS-CoV-2 nsp14 methyltransferase. Nat Commun. 2023;14(1):2259. doi:10.1038/s41467-023-38019-1.

29.

Zhu

Guo

Yan

. Structural insight into the assembly and working mechanism of helicase-primase D5 from Mpox virus. Nat Struct Mol Biol. 2024;31(1):68-81. doi:10.1038/s41594-023-01142-0.

30.

Moss

. Poxvirus DNA replication. Cold Spring Harb Perspect Biol. 2013;5(9):a010199. doi:10.1101/cshperspect.a010199.

31.

Eberhardt

Santos-Martins

Tillack

Forli

. AutoDock Vina 1.2.0: new docking methods, expanded force field, and Python bindings. J Chem Inf Model. 2021;61(8):3891-3898. doi:10.1021/acs.jcim.1c00203.

32.

Lavi

Ngan

Movshovitz-Attias

, et al. Detection of peptide-binding sites on protein surfaces: The first step towards the modeling and targeting of peptide-mediated interactions. Proteins. 2013;81(12):2096-2105. doi:10.1002/prot.24422.

33.

Volkamer

Griewel

Grombacher

Rarey

. Analyzing the topology of active sites: on the prediction of pockets and subpockets. J Chem Inf Model. 2010;50(11):2041-2052. doi:10.1021/ci100241y.

34.

Shannon

Canard

. Nucleotide analogues and mpox: Repurposing the repurposable. Antiviral Res. 2025;234:106057. doi:10.1016/j.antiviral.2024.106057.

35.

Priya

Amalraj

Padmanabhan

, et al. Evaluating the Antiviral Potential of Polyherbal Formulation (Kabasura Kudineer) Against Monkeypox Virus: Targeting E5, Poxin, and DNA Polymerase Through Multifaceted Drug Discovery Approaches. Life. 2025;15(5):771. doi:10.3390/life15050771.

36.

Wallace

Laskowski

Thornton

. LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein Eng. 1995;8(2):127-134. doi:10.1093/protein/8.2.127.

37.

Daina

Michielin

Zoete

. SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci Rep. 2017;7(1):42717. doi:10.1038/srep42717.

38.

Yostawonkul

Kamble

Sakuna

, et al. Effects of Mangosteen (Garcinia mangostana) Peel Extract Loaded in Nanoemulsion on Growth Performance, Immune Response, and Disease Resistance of Nile Tilapia (Oreochromis niloticus) against Aeromonas veronii Infection. Anim Open Access J MDPI. 2023;13(11):1798. doi:10.3390/ani13111798.

39.

Zhou

Wang

Cen

Wei

. Immune regulation and anti-inflammatory effects of isogarcinol extracted from Garcinia mangostana L. against collagen-induced arthritis. J Agric Food Chem. 2014;62(18):4127-4134. doi:10.1021/jf405790q.

40.

Wallert

Bauer

Kluge

, et al. The vitamin E derivative garcinoic acid from Garcinia kola nut seeds attenuates the inflammatory response. Redox Biol. 2019;24:101166. doi:10.1016/j.redox.2019.101166.

41.

Naves

VML

dos Santos

Ribeiro

, et al. Antimicrobial and antioxidant activity of Garcinia brasiliensis extracts. South Afr J Bot. 2019;124:244-250. doi:10.1016/j.sajb.2019.05.021.

42.

Lopes Andrade

Dias Ribeiro Figueiredo

Torequl

, et al. Toxicological evaluation of the biflavonoid, agathisflavone in albino Swiss mice. Biomed Pharmacother Biomedecine Pharmacother. 2019;110:68-73. doi:10.1016/j.biopha.2018.11.050.

43.

Jiang

Liu

Yang

, et al. Flavonolignans and biflavonoids from Cephalotaxus oliveri exert neuroprotective effect via Nrf2/ARE pathway. Phytochemistry. 2022;204:113436. doi:10.1016/j.phytochem.2022.113436.

44.

Onasanwo

Velagapudi

El-Bakoush

Olajide

. Inhibition of neuroinflammation in BV2 microglia by the biflavonoid kolaviron is dependent on the Nrf2/ARE antioxidant protective mechanism. Mol Cell Biochem. 2016;414(1-2):23-36. doi:10.1007/s11010-016-2655-8.

45.

Martin

. A bioavailability score. J Med Chem. 2005;48(9):3164-3170. doi:10.1021/jm0492002.

46.

Wang

Xing

, et al. In silico ADME/T modelling for rational drug design. Q Rev Biophys. 2015;48(4):488-515. doi:10.1017/S0033583515000190.

47.

van de Waterbeemd

Gifford

. ADMET in silico modelling: towards prediction paradise? Nat Rev Drug Discov. 2003;2(3):192-204. doi:10.1038/nrd1032.

48.

Purwaningsih

Maksum

Sumiarsa

Sriwidodo

. Pharmacokinetics and Toxicity Overview of Active Compounds Berberine, Palmatine, and Jatrorrhizine From Fibraurea tinctoria Lour: Drug-Likeness, ADMET Prediction, and In Vivo Extract Toxicity Assessment. J Toxicol. 2025;2025(1):7251602. doi:10.1155/jt/7251602.

49.

Stitz

Schellekens

. Influence of input multiplicity of infection on the antiviral activity of interferon. J Gen Virol. 1980;46(1):205-210. doi:10.1099/0022-1317-46-1-205.

50.

Zembower

Lin

Flavin

Chen

Korba

. Robustaflavone, a potential non-nucleoside anti-hepatitis B agent. Antiviral Res. 1998;39(2):81-88. doi:10.1016/s0166-3542(98)00033-3.

51.

Marchioro

Lde O

De Stefanis

Araújo

, et al. Tetrandrine-driven autophagy suppresses SARS-CoV-2 replication by modulating cholesterol and IGF signaling pathways. Cell Death Discov. 2026;12(1):82. doi:10.1038/s41420-025-02926-7.

52.

Verma

Pratap

. The biological potential of flavones. Nat Prod Rep. 2010;27(11):1571-1593. doi:10.1039/c004698c.

53.

Leonte

Ungureanu

Zaharia

. Flavones and Related Compounds: Synthesis and Biological Activity. Molecules. 2023;28(18):6528. doi:10.3390/molecules28186528.

54.

Doms

Blumenthal

Moss

. Fusion of intra- and extracellular forms of vaccinia virus with the cell membrane. J Virol. 1990;64(10):4884-4892. doi:10.1128/JVI.64.10.4884-4892.1990.

55.

Xing

Wang

, et al. Mpox (formerly monkeypox): pathogenesis, prevention, and treatment. Signal Transduct Target Ther. 2023;8(1):458. doi:10.1038/s41392-023-01675-2.

56.

Selvaraj

Krishnaswamy

Devashya

Sethuraman

Krishnan

. Influence of membrane lipid composition on flavonoid-membrane interactions: Implications on their biological activity. Prog Lipid Res. 2015;58:1-13. doi:10.1016/j.plipres.2014.11.002.

57.

Zlodeeva

Shekunov

Ostroumova

Efimova

. The Degree of Hydroxylation of Phenolic Rings Determines the Ability of Flavonoids and Stilbenes to Inhibit Calcium-Mediated Membrane Fusion. Nutrients. 2023;15(5):1121. doi:10.3390/nu15051121.

58.

Sagdat

Batyrkhan

Kanayeva

. Exploring monkeypox virus proteins and rapid detection techniques. Front Cell Infect Microbiol. 2024;14:1414224. doi:10.3389/fcimb.2024.1414224.

59.

Eilts

Bauer

Fraser

, et al. The diverse role of heparan sulfate and other GAGs in SARS-CoV-2 infections and therapeutics. Carbohydr Polym. 2023;299:120167. doi:10.1016/j.carbpol.2022.120167.

60.

Shi

, et al. SPR Sensor-Based Analysis of the Inhibition of Marine Sulfated Glycans on Interactions between Monkeypox Virus Proteins and Glycosaminoglycans. Mar Drugs. 2023;21(5):264. doi:10.3390/md21050264.

61.

Salles

Meneses

MDF

Yamamoto

, et al. Chemical composition and anti-Mayaro virus activity of Schinus terebinthifolius fruits. Virusdisease. 2021;32(3):526-534. doi:10.1007/s13337-021-00698-z.

62.

Teoh

Sam

, et al. In vitro antiviral activity of Fisetin, Rutin and Naringenin against Dengue virus type-2. J Med Plants Res. 2011;5(23):5534-5539. doi:10.5897/JMPR.9000752.

63.

Rodrigues

Rodrigues Gazolla

da Cruz Pereira

, et al. Synthesis and virucide activity on zika virus of 1,2,3-triazole-containing vanillin derivatives. Antiviral Res. 2023;212:105578. doi:10.1016/j.antiviral.2023.105578.

64.

de Lima

Valente

LMM

Familiar Macedo

, et al. Antiviral and Virucidal Activities of Uncaria tomentosa (Cat’s Claw) against the Chikungunya Virus. Viruses. 2024;16(3):369. doi:10.3390/v16030369.

65.

Khandelwal

Chander

Kumar

, et al. Antiviral activity of Apigenin against buffalopox: Novel mechanistic insights and drug-resistance considerations. Antiviral Res. 2020;181:104870. doi:10.1016/j.antiviral.2020.104870.

66.

Kim

Shin

. A study of flavonoid inhibitors against Monkeypox H1 phosphatase. J Enzyme Inhib Med Chem. 2025;40(1):2535585. doi:10.1080/14756366.2025.2535585.

67.

Dasmahapatra

Kumar

Das

, et al. In-silico molecular modelling, MM/GBSA binding free energy and molecular dynamics simulation study of novel pyrido fused imidazo[4,5-c]quinolines as potential anti-tumor agents. Front Chem. 2022;10:991369. doi:10.3389/fchem.2022.991369.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.65 MB

0.00 MB