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.
In a cytopathic effect inhibition assay, a standardized Rhodiola rosea root and rhizome extract, also known as roseroot extract (SHR-5), exerted distinct anti-influenza A virus activity against HK/68 (H3N2) (IC50 of 2.8 µg/mL) without being cytotoxic. For fast and efficient isolation and identification of the extractʼs bioactive constituents, a high-performance countercurrent chromatographic separation method was developed. It resulted in a three-stage gradient elution program using a mobile phase solvent system composed of ethyl acetate/n-butanol/water (1 : 4 : 5 → 2 : 3 : 5 → 3 : 2 : 5) in the reversed-phase mode. The elaborated high-performance countercurrent chromatographic method allowed for fractionation of the complex roseroot extract in a single chromatographic step in a way that only one additional orthogonal isolation/purification step per fraction yielded 12 isolated constituents. They cover a broad polarity range and belong to different structural classes, namely, the phenylethanoid tyrosol and its glucoside salidroside, the cinnamyl alcohol glycosides rosavin, rosarin, and rosin as well as gallic acid, the cyanogenic glucoside lotaustralin, the monoterpene glucosides rosiridin and kenposide A, and the flavonoids tricin, tricin-5-O-β-D-glucopyranoside, and rhodiosin. The most promising anti-influenza activities were determined for rhodiosin, tricin, and tricin-5-O-β-D-glucopyranoside with IC50 values of 7.9, 13, and 15 µM, respectively. The herein established high-performance countercurrent chromatographic protocol enables fast and scalable access to major as well as minor roseroot constituents. This is of particular relevance for extract standardization, quality control, and further in-depth pharmacological investigations of the metabolites of this popular traditional herbal remedy.
Mulberry Diels−Alder-type adducts (MDAAs) derived from the white mulberry tree were discovered recently as dual inhibitors of influenza viruses and pneumococci. For the development of a natural product based remedy for respiratory infections, the aim was to (i) identify the most prolific natural source of MDAAs, (ii) develop a protocol to maximize the content of MDAAs in Morus alba extracts, (iii) unravel constituents with the highest anti-infective potential within multicomponent mixtures, and (iv) select and characterize a hit extract as a candidate for further studies. Validated quantitative UPLC-PDA analysis of seven MDAAs (1−7) revealed the root bark as the best starting material and pressurized liquid extraction (PLE) as the optimum technique for extraction. Extracts enriched in MDAAs of a total content above 20% exerted a potent dual anti-influenza virus and antipneumococcal activity. For a detailed analysis of the most bioactive chemical features and molecules within the extracts, 1 H NMR-based heterocovariance analysis (HetCA) was used. According to the multivariate statistical analysis procedure conducted, MDAAs exclusively accounted for the in vitro anti-influenza viral effect. The anti-infective profile of one hit extract (MA60) investigated showed a good tolerance by lung cells (A549, Calu-3) and pronounced in vitro activities against influenza viruses, S. pneumoniae, S. aureus, and inflammation.
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