Robust Antiviral Activity of Santonica Flower Extract (Artemisia cina) against Avian and Human Influenza A Viruses: In Vitro and Chemoinformatic Studies

Abstract

The evolution of drug-resistant viral strains following natural acquisition of resistance mutations is a major obstacle to antiviral therapy. Besides the improper prescription of the currently licensed anti-influenza medications, M2-blockers and neuraminidase inhibitors, to control poultry outbreaks/infections potentiates the emergence of drug-resistant influenza variants. Therefore, there is always a necessity to find out new alternatives with potent activity and high safety. Plant extracts and plant-based chemicals represent a historical antiviral resource with remarkable safety in vitro and in vivo to control the emerging and remerging health threats caused by viral infections. Herein, a panel of purified plant extracts and subsequent plant-derived chemicals were evaluated for their anti-avian influenza activity against zoonotic highly pathogenic influenza A/H5N1 virus. Interestingly, santonica flower extract (Artemisia cina) showed the most promising anti-H5N1 activity with a highly safe half-maximal cytotoxic concentration 50 (CC₅₀ > 10 mg/mL) and inhibitory concentration 50 (IC₅₀ of 3.42 μg/mL). To confirm the anti-influenza activity, we assessed the anti-influenza activity of the selected plant extracts against seasonal human influenza A/H1N1 virus and we found that santonica flower extract showed a robust anti-influenza activity that was comparable to the activity against influenza A/H5N1. Furthermore, the mode of action for santonica flower extract with strong inhibitory activity on the abovementioned influenza strains was elucidated, showing a virucidal effect. To go deeper about the activity of the chemometric component of the extract, the major constituent, santonin, was further selected for in vitro screening against influenza A/H5N1 (IC₅₀ = 1.701 μg/mL) and influenza A/H1N1 (IC₅₀ = 2.91 μg/mL). The oxygen of carbonyl functionality in the cyclohexene ring succeeded to form a hydrogen bond with the neuraminidase active site. Despite the fact that santonin revealed similarity to both reference neuraminidase inhibitors in forming hydrogen bonds with essential amino acids, it illustrated shape alignment to oseltamivir more than zanamivir according to Tanimoto algorithms. This study highlights the applicability of santonica flower extract as a promising natural antiviral against low and highly pathogenic influenza A viruses.

Figure 1

Cytotoxicity and anti-H5N1 activities of studied plant extracts in MDCK cells. The CC₅₀ and IC₅₀ of different purified plant extracts were obtained by a crystal violet assay and calculated using nonlinear regression analysis of GraphPad Prism software (version 5.01) by plotting log inhibitor versus normalized response (variable slope).

Figure 2

Cytotoxicity and anti-H1N1 activity of santonica flower extract (a) and oseltamivir carboxylate (b) in MDCK cells. The CC₅₀ and IC₅₀ of santonica flower extract were obtained by a crystal violet assay and calculated using nonlinear regression analysis of GraphPad Prism software (version 5.01) by plotting log inhibitor versus normalized response (variable slope).

Figure 3

Plaque reduction result of santonica flower extract against influenza A/H5N1 (a) and A/H1N1 (b). The viral inhibition (%) was tested in a concentration-dependent manner by a plaque reduction assay and plotted using GraphPad Prism software (version 5.01).

Figure 4

Cytotoxicity and anti-H5N1 (a) and anti-H1N1 (b) activities of santonin in MDCK cells. The CC₅₀ and IC₅₀ of santonin were obtained by a crystal violet assay and calculated using nonlinear regression analysis of GraphPad Prism software (version 5.01) by plotting log inhibitor versus normalized response (variable slope).

Figure 5

Snapshot of selected compounds represented by Vida application: standard ligand (gray color) and its co-crystalized complex (green color) (a); santonin formed an HB with Asn:147A (b); santonin with oseltamivir (c); and santonin with zanamivir (d).

Figure 6

Alignment of santonin with neuraminidase inhibitors. Shape alignment of santonin (green color) with oseltamivir (left) and zanamivir (right) (a). Color shape of santonin (b).