Comparative in vitro analysis of inhibition of rhinovirus and influenza virus replication by mucoactive secretolytic agents and plant extracts - PubMed (original) (raw)

Comparative Study

Comparative in vitro analysis of inhibition of rhinovirus and influenza virus replication by mucoactive secretolytic agents and plant extracts

Christin Walther et al. BMC Complement Med Ther. 2020.

Abstract

Background: Rhinoviruses and influenza viruses cause millions of acute respiratory infections annually. Symptoms of mild acute respiratory infections are commonly treated with over-the-counter products like ambroxol, bromhexine, and N-acetyl cysteine, as well as of thyme and pelargonium extracts today. Because the direct antiviral activity of these over-the-counter products has not been studied in a systematic way, the current study aimed to compare their inhibitory effect against rhinovirus and influenza virus replication in an in vitro setting.

Methods: The cytotoxicity of ambroxol, bromhexine, and N-acetyl cysteine, as well as of thyme and pelargonium extracts was analyzed in Madin Darby canine kidney (MDCK) and HeLa Ohio cells. The antiviral effect of these over-the-counter products was compared by analyzing the dose-dependent inhibition (i) of rhinovirus A2- and B14-induced cytopathic effect in HeLa Ohio cells and (ii) of influenza virus A/Hong Kong/68 (subtype H3N2)- and A/Jena/8178/09 (subtype H1N1, pandemic)-induced cytopathic effect in MDCK cells at non-cytotoxic concentrations. To get insights into the mechanism of action of pelargonium extract against influenza virus, we performed time-of-addition assays as well as hemagglutination and neuraminidase inhibition assays.

Results: N-acetyl cysteine, thyme and pelargonium extract showed no or only marginal cytotoxicity in MDCK and HeLa Ohio cells in the tested concentration range. The 50% cytotoxic concentration of ambroxol and bromhexine was 51.85 and 61.24 μM, respectively. No anti-rhinoviral activity was detected at non-cytotoxic concentrations in this in vitro study setting. Ambroxol, bromhexine, and N-acetyl cysteine inhibited the influenza virus-induced cytopathic effect in MDCK cells no or less than 50%. In contrast, a dose-dependent anti-influenza virus activity of thyme and pelargonium extracts was demonstrated. The time-of addition assays revealed an inhibition of early and late steps of influenza virus replication by pelargonium extract whereas zanamivir acted on late steps only. The proven block of viral neuraminidase activity might explain the inhibition of influenza virus replication when added after viral adsorption.

Conclusion: The study results indicate a distinct inhibition of influenza A virus replication by thyme and pelargonium extract which might contribute to the beneficial effects of these plant extracts on acute respiratory infections symptoms.

Keywords: Acute respiratory infection; Ambroxol; Antiviral; Bromhexine; Mechanism; N-acetyl cysteine; Natural products; Over-the-counter medication; Pelargonium; Thyme; Virus.

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Conflict of interest statement

The authors declare that they have no competing interest.

Figures

Fig. 1

Fig. 1

Dose-dependent cytotoxicity and anti-rhinovirus activity of pleconaril and test items in HeLa Ohio cells. The cytotoxicity and anti-influenza activity of zanamivir (a), N-acetylcystein (b), bromhexine (c), ambroxol (d), thyme extract (e), and pelargonium extract (f) was analyzed in MDCK cells. The percentage of cell viability was used as readout for analyzing the cytotoxic and antiviral effects. Cell viability was evaluated as the percentage of the mean value of optical density resulting from 6 cell controls, which was set 100%. Cytotoxic compounds have the potential to reduce cell viability. Influenza virus A/Hong Kong/68 (HK/68) and influenza virus A/Jena/8178/09 (Jena/8178) were included in the antiviral studies. Replication of both viruses results in a complete cytopathic effect of untreated MDCK cells thereby also reducing cell viability. Effective compound/extract treatment enhances the percentage of cell viability. Means and standard deviations of at least three independent experiments are shown

Fig. 2

Fig. 2

Dose-dependent cytotoxicity and anti-influenza virus activity of zanamivir and test items in MDCK cells. The cytotoxicity and anti-influenza virus activity of zanamivir (a), N-acetylcystein (b), bromhexine (c), ambroxol (d), thyme extract (e), and pelargonium extract (f) was analyzed in MDCK cells. The percentage of cell viability was used as readout for analyzing cytotoxic and antiviral effects. Cell viability was evaluated as the percentage of the mean value of optical density resulting from 6 cell controls, which was set 100%. Cytotoxic compounds have the potential to reduce cell viability. Influenza virus A/Hong Kong/68 (HK/68) and influenza virus A/Jena/8178/09 (Jena/8178) were included in the antiviral studies. Replication of both viruses results in a complete cytopathic effect of untreated MDCK cells thereby reducing cell viability. Effective compound/extract treatment enhances the percentage of cell viability. Means and standard deviations of at least three independent experiments are shown

Fig. 3

Fig. 3

Pelargonium extract affects various steps of the replication cycle of influenza virus A/Hong Kong/68. We performed plaque reduction assays with influenza virus A/Hong Kong/68 and pelargonium extract (50 μg/mL) in MDCK cells. Zanamivir (1 μM) was included as reference inhibitor. We added pelargonium extract or zanamivir to MDCK cells and/or influenza virus A/Hong Kong/68 at different times before virus infection (pretreatment of cells or virus; at 37 °C for 1 h), during virus adsorption (at 4 °C for 2 h), after virus adsorption (at 37 °C for about 72 in the agar overlay), and during as well as after adsorption. After an incubation at 37 °C for 72 h and crystal violet staining, we counted the plaques over a light box. The mean plaque number of three mock-treated virus controls was set to 100%. The bars indicate the mean and standard deviation of the percent plaque reduction by pelargonium extract and zanamivir compared to the mock-treated virus control. Means and standard deviations of three independent experiments are shown

Fig. 4

Fig. 4

Representative photographs visualizing the effects of pelargonium extract on virus-mediated hemagglutination and viral neuraminidase activity. a Effect on hemagglutination: Four hemagglutination units of influenza virus A/Jena/8178/2009 (8178/09: +) or phosphate-buffered saline (8178/09: -) were mixed with phosphate-buffered saline or with serial half-logarithmic dilutions of zanamivir (1 to 0.0003 nM) or pelargonium extract (100 to 0.03 μg/mL). Then we added a human erythrocyte solution at 4° for 2 h. No hemagglutination occurs in the absence of virus in control 1 (Co1). Control 2 shows the virus-mediated hemagglutination of mock-treated human erythrocytes (Co2). Pelargonium extract induced a hemagglutination at 3 and 10 μg/mL and blocked the influenza virus-mediated hemagglutination at 30 and 100 μg/mL. b Inhibition of viral neuraminidase activity: After protocolling the effect on hemagglutination, we further incubated the test at 37 °C overnight. In the absence of inhibitors, the activated viral neuraminidase abolished the virus-mediated hemagglutination as seen in Co2. Both zanamivir and pelargonium extract blocked the neuraminidase activity with minimal inhibitory concentrations of 0.03 μM and 0.3 μg/mL, respectively. We repeated the experiments once to confirm the inhibitory effects

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