Integrated In Silico, In Vitro, and In Vivo Studies Reveal Mangiferin as a Promising Antiviral Agent Against H1N1/pdm2009 Influenza Virus

Abstract

The ongoing global threat posed by the influenza A virus, exacerbated by antigenic drift and the emergence of antiviral resistance, accentuates the urgent need for innovative therapeutic strategies. Through molecular docking, this study revealed that mangiferin has a strong binding affinity for the active site of the neuraminidase (NA) protein of influenza virus A(H1N1)pdm09, with a binding energy of -8.1 kcal/mol. In vitro assays confirmed a dose-dependent inhibition of NA, with an IC₅₀ of 88.65 μM, and minimal cytotoxicity, as indicated by a CC₅₀ of 328.1 μM in MDCK cells. In murine models, the administration of mangiferin at a dosage of 25 mg/kg significantly mitigated weight loss, decreased viral loads in nasal turbinates and lungs by over 1 log10 TCID₅₀, and enhanced survival rates from 0% in control groups to 20% in mangiferin-treated group at 14 days post-infection. In addition, mangiferin was found to modulate host immune responses by simultaneously inhibiting pro-inflammatory cytokines, IL-6 and TNF-α, and upregulating the expression of anti-inflammatory IL-10 and antiviral IFN-γ, thus mitigating infection-induced inflammation. Our findings elucidate the dual mechanism of mangiferin involving the direct inhibition of NA and immunomodulation, thereby providing experimental evidence for exploring dual-mechanism-based anti-influenza strategies against resistant strains of influenza.

Keywords: influenza virus A(H1N1)pdm09; mangiferin; molecular docking.

Figure 1

Interaction of mangiferin with A(H1N1)pdm09 neuraminidase protein revealed through molecular docking. (A) Mangiferin (red) bound neuraminidase (silver) at its active binding site. (B) Binding mode of mangiferin with neuraminidase revealing the interacting amino acid residues and bonds at the active binding site of neuraminidase.

Figure 2

Mangiferin’s anti-neuraminidase activity and cytotoxicity in vitro. (A) Dose–response curve of mangiferin on neuraminidase inhibition (IC₅₀ = 88.65 μM; R² = 0.9898). (B) Evaluation of mangiferin cytotoxicity using the MTT assay (R² = 0.8426). (C) Viral titers of influenza virus A(H1N1)pdm09 treated with different mangiferin concentrations (Student’s t-test was used to assess statistical significance, compared to 0 μM; * indicates p < 0.05, ** indicates p < 0.01; **** indicates p < 0.0001).

Figure 3

Clinical observations across different experimental groups of mice. (A) Changes in body weight using two-way ANOVA. (B) Survival rates by log-rank test (χ² = 20.51, p < 0.0001), with a significant trend across groups (p = 0.0018). (C) Viral titer in nasal turbinates. (D) Viral titer in lungs. In (C,D), statistical significance was determined by unpaired multiple t-tests. * p < 0.05; *** p < 0.001.

Figure 4

The relative expression levels of cytokines IL-6, IL-10, TNF-α, and IFN-γ in mice lung tissues across the three experimental mice groups—the control group (uninfected and untreated mice), the H1N1-infected group, and the H1N1-infected group treated with mangiferin. (A) IL-6; (B) IL-10; (C) TNF-α; (D) IFN-γ. Statistical significance was determined by s two-way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.