Multi-Target Inhibition of Influenza A Viral Proteins by Medicinal Plant Phytochemicals: Integrated Molecular Docking and In-Vitro Antiviral Validation

Influenza A virus infection remains a persistent global public health concern responsible for seasonal epidemics and occasional pandemics associated with significant morbidity and mortality. Continuous antigenic drift and shift mechanisms enable rapid viral evolution, allowing influenza viruses to escape host immune responses and reduce the long-term effectiveness of available antiviral therapies. Currently approved antiviral drugs, including M2 ion channel inhibitors and neuraminidase inhibitors, have shown declining clinical efficacy due to the emergence of resistant viral strains, thereby emphasizing the urgent need for alternative therapeutic strategies targeting multiple stages of viral replication¹,². Recent advances in antiviral research have highlighted medicinal plant–derived phytochemicals as promising candidates for novel drug discovery. Natural compounds possess extensive structural diversity and biological compatibility, enabling interaction with a wide range of viral targets while often demonstrating reduced toxicity profiles. Several classes of phytochemicals, including flavonoids, polyphenols, and terpenoid derivatives, have demonstrated inhibitory activity against respiratory viruses through interference with viral entry, replication, and release mechanisms³.

 

Influenza viral proteins such as matrix protein-2 (M2), neuraminidase (NA), and hemagglutinin (HA) play essential roles in the viral life cycle. The M2 ion channel regulates proton transport required for viral uncoating, neuraminidase facilitates release of newly formed viral particles from infected host cells, and hemagglutinin mediates viral attachment and membrane fusion during host cell entry. Targeting these proteins simultaneously represents an effective strategy to suppress viral propagation and reduce the likelihood of resistance development⁴,⁵. Computational approaches such as molecular docking have become valuable tools in modern antiviral drug discovery by enabling rapid prediction of ligand–protein interactions prior to experimental validation. Docking-based virtual screening allows prioritization of bioactive compounds with favorable binding characteristics, thereby reducing experimental workload and accelerating lead identification⁶,⁷. However, reliance solely on computational prediction may not accurately reflect biological activity, highlighting the importance of combining in-silico analysis with laboratory-based validation studies⁸.

 

Although several investigations have explored phytochemical interactions with individual influenza viral proteins, comprehensive studies integrating multi-target docking analysis with experimental antiviral confirmation remain limited. Many reported studies focus exclusively on computational screening without validating antiviral efficacy under biological conditions⁹. Consequently, identification of phytochemicals capable of simultaneously inhibiting multiple influenza targets with confirmed in-vitro activity remains an important research requirement. Therefore, the present study was designed to evaluate selected medicinal plant phytochemicals against three critical influenza A viral proteins M2, neuraminidase, and hemagglutinin using an integrated computational and experimental framework. The study hypothesizes that plant-derived phytochemicals demonstrating strong multi-target binding affinity will exhibit measurable antiviral activity during in-vitro evaluation. This combined strategy aims to support the discovery of multi-target natural antiviral leads capable of overcoming drug resistance and contributing to future influenza therapeutic development¹⁰.

 

Molecular Docking Analysis of Phytochemicals Against Influenza Viral Proteins

Molecular docking analysis was performed to evaluate the interaction potential of selected phytochemicals with three essential influenza A viral proteins, namely matrix protein-2 (M2), neuraminidase (NA), and hemagglutinin (HA). All evaluated compounds demonstrated measurable binding affinity toward at least one viral target protein. Docking energy values ranged from −7.0 to −9.5 kcal/mol, indicating favorable ligand–protein interactions consistent with potential antiviral activity. Comparative evaluation identified several phytochemicals capable of interacting efficiently with multiple viral targets. Among the tested compounds, epigallocatechin gallate (EGCG), curcumin, baicalein, and luteolin demonstrated consistently strong binding energies across all three viral proteins, suggesting multi-target inhibitory capability. EGCG exhibited the strongest interaction particularly with neuraminidase and hemagglutinin proteins, indicating stable binding within functional regions of viral activity.

 

Table 1. Docking binding energies of phytochemicals against influenza viral proteins

Compound	M2 (kcal/mol)	NA (kcal/mol)	HA (kcal/mol)	Normalized Docking Score
Quercetin	−7.8	−8.6	−7.9	0.88
Kaempferol	−7.3	−8.1	−7.5	0.84
Luteolin	−8.1	−8.9	−8.2	0.91
Apigenin	−7.0	−7.8	−7.2	0.80
Curcumin	−8.5	−9.2	−8.6	0.94
EGCG	−8.9	−9.5	−9.1	0.97
Glycyrrhizin	−7.6	−8.4	−7.8	0.86
Berberine	−8.0	−8.7	−8.1	0.89
Rosmarinic acid	−7.4	−8.0	−7.6	0.83
Baicalein	−8.3	−9.0	−8.4	0.93

 

 

Figure – 1. Illustrates comparative docking performance of phytochemicals across M2, NA, and HA proteins using heatmap visualization.

 

Interaction Characteristics and Multi-Target Binding Potential

Analysis of docking conformations revealed that high-ranking phytochemicals occupied functionally significant regions within each viral protein structure. Strong binding compounds were positioned within the M2 ion channel lumen, suggesting potential interference with proton transport mechanisms. Within neuraminidase, leading compounds demonstrated stable positioning inside catalytic pockets, indicating possible inhibition of viral particle release. Interaction analysis for hemagglutinin showed ligand binding near receptor-binding domains responsible for host cell attachment. Normalized docking score comparison identified EGCG, curcumin, baicalein, and luteolin as compounds exhibiting balanced interaction strength across all viral targets, supporting their classification as multi-target inhibitors.

 

In-Vitro Antiviral Activity

Biological validation using plaque reduction assays demonstrated dose-dependent inhibition of influenza A viral replication for all selected phytochemicals. Increasing compound concentration resulted in progressive reduction of viral plaque formation. Observed IC₅₀ values ranged between 9.6 and 25.4 µM, indicating measurable antiviral effectiveness. EGCG showed the strongest antiviral activity followed by curcumin, baicalein, and luteolin, which displayed comparatively lower IC₅₀ values and higher viral inhibition efficiency.

 

Table 2. In-vitro antiviral efficacy and cytotoxicity profile

Compound	IC₅₀ (µM)	CC₅₀ (µM)	Selectivity Index	Plaque Reduction (%)	Cytotoxicity (%)
Quercetin	18.5	220	11.9	72	8
Kaempferol	21.2	210	9.9	68	10
Luteolin	14.6	240	16.4	78	7
Apigenin	25.4	195	7.7	61	12
Curcumin	11.8	260	22.0	85	6
EGCG	9.6	280	29.1	90	5
Glycyrrhizin	19.7	230	11.7	70	9
Berberine	15.9	215	13.5	76	8
Rosmarinic acid	22.3	205	9.2	65	11
Baicalein	12.9	250	19.4	82	6

 

Figure – 2. Presents dose-response curves demonstrating inhibition of influenza viral replication by selected phytochemicals.

Cytotoxicity and Selectivity Evaluation

Cytotoxicity assessment indicated acceptable safety profiles for all evaluated compounds within tested concentration ranges. CC₅₀ values exceeded 195 µM for all phytochemicals, indicating minimal adverse effects on host cell viability. EGCG and curcumin demonstrated the highest selectivity index values, reflecting an optimal balance between antiviral potency and cellular safety. Cytotoxicity levels remained below 12% at 50 µM concentration across all tested compounds.

 

Correlation Between Computational Prediction and Experimental Activity

Correlation analysis demonstrated a significant inverse relationship between docking binding energies and experimentally determined IC₅₀ values. Compounds exhibiting stronger predicted binding affinity generally showed enhanced antiviral activity during in-vitro evaluation. The calculated Pearson correlation coefficient indicated strong agreement between computational screening results and biological validation outcomes.

 

Figure – 3. shows correlation between normalized docking scores and antiviral potency

The continuous emergence of drug-resistant influenza strains represents a major challenge to current antiviral treatment strategies. Conventional antiviral agents typically target a single viral protein, which facilitates rapid development of resistance through viral mutation. The present investigation explored an alternative multi-target therapeutic approach by evaluating medicinal plant–derived phytochemicals capable of simultaneously interacting with multiple influenza A viral proteins. The integrated computational and experimental findings demonstrate that several phytochemicals possess promising antiviral potential through coordinated inhibition of M2, neuraminidase, and hemagglutinin proteins.

 

Molecular docking analysis revealed strong interaction of selected phytochemicals within functionally critical regions of viral proteins. Binding within the M2 ion channel suggests possible disruption of proton transport required for viral uncoating, whereas interactions observed in neuraminidase catalytic pockets indicate potential inhibition of viral particle release from infected host cells. Similarly, ligand positioning near hemagglutinin receptor-binding domains may interfere with viral attachment and membrane fusion processes. Structural studies have previously emphasized that simultaneous inhibition of viral entry, replication, and release pathways significantly enhances antiviral effectiveness compared with single-target inhibition strategies¹⁹,²⁰.

 

Among the tested compounds, epigallocatechin gallate, curcumin, baicalein, and luteolin demonstrated consistent binding affinity across all evaluated viral targets. Polyphenolic phytochemicals are known to exhibit antiviral activity through multiple molecular mechanisms, including protein binding, enzyme inhibition, and modulation of host cellular responses. Earlier investigations have reported broad-spectrum antiviral properties of flavonoids and related compounds against influenza viruses, supporting their suitability as multi-target therapeutic candidates²¹,²².

 

In-vitro validation further confirmed the biological relevance of computational predictions. The observed dose-dependent reduction in viral replication indicates that docking-derived binding affinity translated into measurable antiviral activity under experimental conditions. Compounds exhibiting stronger docking interactions demonstrated lower IC₅₀ values, supporting the predictive capability of molecular docking approaches when combined with biological validation. Similar correlations between computational screening and experimental antiviral outcomes have been reported in modern antiviral drug discovery workflows²³.

 

An important outcome of the present study is the demonstrated advantage of integrating in-silico screening with in-vitro confirmation. Computational methods alone may overlook pharmacological complexity arising from cellular uptake, metabolic stability, or off-target effects. Experimental validation therefore remains essential to confirm functional antiviral activity. Integrated workflows combining virtual screening and laboratory testing have increasingly been recommended for efficient identification of natural antiviral leads while minimizing experimental cost and time requirements²⁴.

 

Cytotoxicity evaluation indicated favorable safety profiles for the investigated phytochemicals, with high selectivity index values observed particularly for EGCG and curcumin. A high selectivity index reflects effective viral inhibition with minimal host cell toxicity, which represents an important requirement for antiviral drug development. Natural compounds with balanced efficacy and safety profiles are increasingly recognized as valuable starting points for development of next-generation antiviral therapeutics²². The multi-target inhibitory nature of identified phytochemicals may also contribute to reduced probability of resistance development. Influenza viruses frequently acquire resistance mutations when exposed to drugs acting on single molecular targets. Polypharmacological agents capable of simultaneously affecting multiple viral proteins create higher evolutionary barriers for resistance emergence, an approach increasingly emphasized in antiviral research and drug repurposing strategies²¹²⁵.

 

Despite encouraging findings, certain limitations should be acknowledged. The study relied on docking-based interaction prediction without incorporation of molecular dynamics simulations to evaluate long-term binding stability. Additionally, antiviral activity was evaluated under controlled in-vitro conditions, which may not fully represent complex host immune interactions observed in vivo. Future investigations involving mechanistic studies, pharmacokinetic evaluation, and animal model validation are required to establish therapeutic applicability.

 

Overall, the present study highlights the potential of medicinal plant phytochemicals as multi-target anti-influenza agents and demonstrates that integration of computational modeling with experimental validation provides a reliable framework for early-stage antiviral drug discovery.

The present study successfully demonstrated the antiviral potential of selected medicinal plant–derived phytochemicals against key influenza A viral proteins through an integrated molecular docking and in-vitro validation approach. Computational analysis identified several phytochemicals capable of strong interaction with matrix protein-2, neuraminidase, and hemagglutinin, indicating effective multi-target inhibitory capability.

Experimental validation confirmed that compounds such as epigallocatechin gallate, curcumin, baicalein, and luteolin significantly inhibited influenza viral replication in a dose-dependent manner while maintaining low cytotoxic effects on host cells. The observed correlation between docking predictions and biological antiviral activity supports the reliability of computational screening as an early-stage tool in antiviral drug discovery.

 

The ability of these phytochemicals to simultaneously target multiple viral proteins represents an important advantage over conventional single-target antivirals and may contribute to reduced emergence of drug resistance. Integration of computational modeling with biological validation therefore provides an efficient framework for identification of plant-based antiviral lead molecules.

 

Future research should focus on molecular dynamics simulations, mechanistic pathway analysis, pharmacokinetic evaluation, and in-vivo validation to further establish therapeutic applicability. Overall, the findings provide a scientific basis for the development of multi-target natural antiviral agents for influenza management.

 

 

Acknowledgement

The authors express sincere gratitude to SSV Bioscience PVT LTD., Erode, Tamil Nadu for providing scientific guidance, research support, and facilities during the execution of this postgraduate dissertation research work.

 

Conflict of Interest

The authors declare that there is no conflict of interest related to this study.