Anti-infective Activities of Pelargonium sidoides (EPS® 7630): Effects of Induced NO Production on Leishmania major in Infected Macrophages and Antiviral Effects as Assessed in a Fibroblast-Virus Protection Assay

Abstract

EPs® 7630 is an aqueous-ethanolic extract of the roots of Pelargonium sidoides, employed in the treatment of upper respiratory tract infections. Its anti-infective activity is supposed to be associated with the activation of the nonspecific immune system. Using Leishmania major GFP-infected murine BMMΦ, the NO production of EPs® 7630-activated macrophages was correlated with the reduction of the GFP signal measured at single cell levels using flow cytometry. The anti-infectious effect of EPs® 7630 (3–10 µg/mL) on its own (NO production: 4–13 µM; signal reduction: 25–73 %) was less prominent than that in combination with IFN-γ (100 U/mL) (NO production: 20–27 µM; signal reduction: 35–78 %). Furthermore, supernatants of EPs® 7630-stimulated BMMΦ (10 µg/mL) significantly reduced the cytopathic effect of EMCV on L929 fibroblasts (antiviral activity 80 U/mL) when compared with an IFN-γ standard (100 U/mL). Direct addition of EPs® 7630 to L929 did not mediate cytoprotective effects. The antiviral components induced in BMMΦ by EPs® 7630 remain to be identified. Detection of any IFNs by ELISA was unsuccessful, which may be due to their very low concentrations in cell supernatants. The current data provide convincing support for the induction of anti-infectious responses by EPs® 7630.

Key words

Pelargonium sidoides - Geraniaceae - anti‐infective - nitric oxides - Leishmania major GFP - antiviral - encephalomyocarditis virus

References

• 1 Matthys H, Eisebitt R, Seith B, Heger M. Efficacy and safety of an extract of Pelargonium sidoides (EPs® 7630) in adults with acute bronchitis.  Phytomedicine. 2003;  10 (Suppl. 4) 7-17
• 2 Haidvogl M, Heger M. Treatment effect and safety of EPs® 7630-solution in acute bronchitis in childhood: report of a multicentre observational study.  Phytomedicine. 2007;  14 (Suppl. 1) 60-64
• 3 Matthys H, Heger M. EPs® 7630-solution – an effective therapeutic option in acute and exacerbating bronchitis.  Phytomedicine. 2007;  14 (Suppl. 1) 65-68
• 4 Schulz V. Liquid herbal drug preparation from the root of Pelargonium sidoides is effective against acute bronchitis: results of a double-blind study with 124 patients.  Phytomedicine. 2007;  14 (Suppl. 6) 74-75
• 5 Matthys H, Funk P. EPs® 7630 improves acute bronchitic symptoms and shortens time to remission. Results of a randomised, double-blind, placebo-controlled, multicentre trial.  Planta Med. 2008;  74 686-692
• 6 Agbabiaka T B, Guo R, Ernst E. Pelargonium sidoides for acute bronchitis: a systematic review and meta-analysis.  Phytomedicine. 2008;  15 378-385
• 7 Thäle C, Kiderlen A, Kolodziej H. Anti-infective mode of action of EPs® 7630 at the molecular level.  Planta Med. 2008;  74 675-681
• 8 Kram D, Thäle C, Kolodziej H, Kiderlen A F. Intracellular parasite kill: flow cytometry and NO detection for rapid discrimination between anti-leishmanial activity and macrophage activation.  J Immunol Methods. 2008;  333 79-88
• 9 Kolodziej H, Kiderlen A F. In vitro evaluation of antibacterial and immunomodulatory activities of Pelargonium reniforme, Pelargonium sidoides and the related herbal drug preparation EPs® 7630.  Phytomedicine. 2007;  14 (Suppl. 6) 18-26
• 10 Schoetz K, Erdelmeier C, Germer S, Hauer H. A detailed view on the constituents of EPs® 7630.  Planta Med. 2008;  74 667-674
• 11 Ha D S, Schwarz J K, Turco S J, Beverley S M. Use of the green fluorescent protein as a marker in transfected Leishmania.  Mol Biochem Parasitol. 1996;  77 57-64
• 12 Berens R L, Marr J J. An easily prepared defined medium for cultivation of Leishmania donovani promastigotes.  J Parasitol. 1978;  64 160
• 13 Kiderlen A F, Kaye P M. A modified colorimetric assay of macrophage activation for intracellular cytotoxicity against Leishmania parasites.  J Immunol Methods. 1990;  127 11-18
• 14 Sasaki D T, Dumas S E, Engleman E G. Discrimination of viable and non-viable cells using propidium iodide in two color immunofluorescence.  Cytometry. 1987;  8 413-420
• 15 Meager A. Assays for antiviral activity.  Methods Mol Biol. 2004;  249 121-134
• 16 Ding A H, Nathan C F, Stuehr D J. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production.  J Immunol. 1988;  141 2407-2412
• 17 Green S J, Meltzer M S, Hibbs Jr J B, Nacy C A. Activated macrophages destroy intracellular Leishmania major amastigotes by an L-arginine-dependent killing mechanism.  J Immunol. 1990;  144 278-283
• 18 Lemesre J L, Sereno D, Daulouède S, Veyret B, Brajon N, Vincendeau P. Leishmania spp.: nitric oxide-mediated metabolic inhibition of promastigote and axenically grown amastigote form.  Exp Parasitol. 1997;  86 58
• 19 Awasthi A R, Mathur K, Saha B. Immune response to Leishmania infection.  Indian J Med Res. 2004;  119 238-258
• 20 Schroder K P, Hertzog J, Ravasi T, Hume D A. Interferon-gamma: an overview of signals, mechanisms and functions.  J Leukoc Biol. 2004;  75 163-189
• 21 Mosser D M. The many faces of macrophage activation.  J Leukoc Biol. 2003;  73 209-212
• 22 Herwaldt B L. Leishmaniasis.  Lancet. 1999;  354 1191-1199
• 23 Kolodziej H, Radtke O A, Kiderlen A F. Stimulus (polyphenol, IFN-gamma, LPS)-dependent nitric oxide production and antileishmanial effects in RAW 264.7 macrophages.  Phytochemistry. 2008;  69 3103-3110
• 24 Sakuma I, Stuehr D J, Gross S S, Nathan C, Levi R. Identification of arginine as a precursor of endothelium-derived relaxing factor.  Proc Natl Acad Sci USA. 1988;  85 8664-8667
• 25 Schroder K, Sweet M J, Hume D A. Signal integration between IFNgamma and TLR signalling pathways in macrophages.  Immunobiology. 2006;  211 511-524
• 26 Fujihara M, Muroi M, Tanamoto K, Suzuki T, Azuma H, Ikeda H. Molecular mechanisms of macrophage activation and deactivation by lipopolysaccharide: roles of the receptor complex.  Pharmacol Ther. 2003;  100 171-194
• 27 Doherty T M, Sher A, Vogel S N. Paclitaxel (Taxol)-induced killing of Leishmania major in murine macrophages.  Infect Immun. 1998;  66 4553-4556
• 28 Perera P Y, Mayadas T N, Takeuchi O, Akira S, Zaks-Zilberman M, Goyert S M, Vogel S N. CD11b/CD18 acts in concert with CD14 and Toll-like receptor (TLR) 4 to elicit full lipopolysaccharide and taxol-inducible gene expression.  J Immunol. 2001;  166 574-581
• 29 Vadiveloo P K, Vairo G, Hertzog P, Kola I, Hamilton J A. Role of type I interferons during macrophage activation by lipopolysaccharide.  Cytokine. 2000;  12 1639-1646
• 30 Toshchakov V, Jones B W, Perera P Y, Thomas K, Cody M J, Zhang S, Williams B R, Major J, Hamilton T A, Fenton M J, Vogel S N. TLR4, but not TLR2, mediates IFN-beta-induced STAT1alpha/beta-dependent gene expression in macrophages.  Nat Immunol. 2002;  3 392-398
• 31 MacMicking J, Xie Q W, Nathan C. Nitric oxide and macrophage function.  Annu Rev Immunol. 1997;  15 323-350
• 32 PBL Interferon Source .Cyotopathic effect assay 101. Available at. http://www.biocompare.com/Articles/ApplicationNote/1664/Cytopathic-Effect-Assay-101.html Accessed July 13, 2010
• 33 Ank N, Iversen M B, Bartholdy C, Staeheli P, Hartmann R, Jensen U B, gnaes-Hansen F, Thomsen A R, Chen Z, Haugen H, Klucher K, Paludan S R. An important role for type III interferon (IFN-lambda/IL-28) in TLR-induced antiviral activity.  J Immunol. 2008;  180 2474-2485
• 34 Tafalla C, Figueras A, Novoa B. Possible role of LTB₄ in the antiviral activity of turbot (Scophthalmus maximus) leukocyte-derived supernatants against viral hemorrhagic septicaemia virus (VHSV).  Dev Comp Immunol. 2002;  26 283-293
• 35 Gaudreault E, Gosselin J. Leukotriene B₄ induces release of antimicrobial peptides in lungs of virally infected mice.  J Immunol. 2008;  180 6211-6221
• 36 Koch E, Augustin R, Wohn C, Erdelmeier C A J. Inhibition of mediator release from RBL-2H3 cells and human granulocytes by the Pelargonium sidoides root extract EPs® 7630.  Planta Med. 2010;  76 1291