In vitro antiviral activities of thymol and Limonin against influenza a viruses and SARS-CoV-2

Abstract

Emerging and re-emerging respiratory viruses represent a continuing threat to human health. The pandemic severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and influenza A viruses (IAVs) are co-circulating, presenting serious threats to public health. Therefore, screening for safe and broad-spectrum antiviral candidates to control such viral infections is prioritized. Herein, this study reports the in vitro antiviral activity of some essential volatile oils (EOs) and volatile oil components including Peppermint oil, Eucalyptus oil, Clove oil, Thymol, Camphor and Limonin against two different IAVs, namely influenza A/H1N1 and A/H5N1 viruses, and SARS-CoV-2 virus. All tested samples were safe in MDCK and Vero E6 cell lines with CC₅₀ values that exceed 1 mg/ml, allowing the screening of their antiviral activities using a wide range of concentrations. The results show the potency of Thymol and Limonin against influenza A/H1N1 virus with IC₅₀ values of 0.022 and 4.25 µg/ml, respectively. The anti-influenza activities of Thymol and Limonin were further validated by testing them against the avian influenza A/H5N1 virus, resulting in anti-influenza activities with IC₅₀ values of 18.5 and 15.6 ng/ml, respectively. The broad-spectrum potential of the highly potent antiviral candidates, Thymol and Limonin, were further tested against the pandemic SARS-CoV-2 and, both exerted anti-coronavirus activities with IC₅₀ values of 0.591 and 4.04 µg/ml, respectively. Further investigations against influenza A/H1N1 virus revealed that Thymol and Limonin could inhibit IAV by hindering viral replication. The Biochemical analyses of the interaction of Limonin and Thymol with FDA-approved anti-influenza drug targets, neuraminidase and viral polymerases, revealed that both compounds can partially inhibit IAV polymerase activity, but have no effect on neuraminidase activity. Likely, molecular docking studies indicated that Thymol and Limonin obstruct active binding sites of IAV polymerases. These findings presented on the antiviral activity of Limonin and Thymol might be used to support the development of supplemental therapy against currently emerging and reemerging respiratory viral infections.

Keywords: Essential oils; IAV; Limonin; Respiratory viruses; SARS-CoV-2; Thymol.

Fig. 1

Cytotoxicity of the investigated essential oils (EOs) and EOs components. (a) Cytotoxicity of the investigated EOs on the MDCK cell line; (b) Cytotoxicity of Thymol and Limonin in Vero E6 cells. By plotting log inhibitor versus normalized response (variable slope) and running non-linear regression analyses using GraphPad Prism 5.01 software, the CC₅₀ values were determined.

Fig. 2

Antiviral activities of the investigated essential oils (EOs) and EOs components. (a) Antiviral effects of the investigated EOs and EOs components against seasonal human influenza A/H1N1 virus; (b) Antiviral effects of Thymol and Limonin against highly pathogenic avian influenza A/H5N1 virus. (c) Anti-coronavirus activity against SARS-CoV-2. By plotting log inhibitor versus normalized response (variable slope) and running non-linear regression analyses using GraphPad Prism 5.01 software, the IC₅₀ of the investigated EOs were determined.

Fig. 3

Viral inhibition after treatment with two different concentrations of the highly promising essential oils as measured by plaque reduction assay. The viral reduction in influenza A/H1N1 and A/H5N1 following treatment with the two essential oils were depicted in percentages. Each oil was evaluated independently against both viruses and data were plotted using GraphPad Prism 5.01 software.

Fig. 4

Biochemical assays to explore the anti-polymerase and anti-neuraminidase activities of Limonin and Thymol. (a) Minigenome assay to validate possible biological interaction of Thymol and Limonin with influenza A/H1N1 polymerase subunits as determined by ZsGreen expression in treated and untreated transfected HEK 293T cells or (b) by measuring the nanoluciferase activity in the supernatants of treated and untreated transfected HEK 293T cells. (c) The neuraminidase inhibition assay against influenza A/H1N1 showed that Limonin and Thymol have a rare effect on viral neuraminidase activity.

Fig. 5

(a) Superimposition of the co-crystallized ligand (Pimodivir) of IAV PB2 (carbon atoms in green) and the docked pose of the same ligand (carbon atoms in turquoise); (b) Superimposition of the co-crystallized ligand (Baloxavir acid) of IAV PA (carbon atoms in green) and the docked pose of the same ligand (carbon atoms in violet).

Fig. 6

(a) Binding mode of the co-crystallized ligand (Pimodivir) against IAV PB2 protein; (b) Binding mode of Thymol against IAV PB2 protein; (c) Binding mode of Limonin against IAV PB2 protein.

Fig. 7

(a) Binding mode of the co-crystallized ligand (Baloxavir acid) with IAV PA protein; (b) Binding mode of Thymol IAV PA protein; (c) Binding mode of Limonin with IAV PA protein.