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Extract of Pelargonium sidoides (EPs[R] 7630) improves phagocytosis, oxidative burst, and intracellular killing of human peripheral blood phagocytes in vitro.

Abstract

Clinical data show that EPs[R] 7630, an aqueous ethanolic extract from the roots of Pelargonium sidoides, can be used for the treatment of upper respiratory tract infections (URTI). The biological effects of the preparation have not been fully investigated. The objective of this study was to examine the impact of EPs[R] 7630 on the activity of human peripheral blood phagocytes (PBP).

A whole blood-based, flow cytometric assay was used to simultaneously assess phagocytosis and oxidative burst. Calcein-AM stained Candida albicans (DSM 1386) were used as target organisms. Oxidative burst was measured by addition of dihydroethidium (DHE). Target organisms and whole blood were co-incubated and analyzed after 0, 2, 4, 6, 10, and 30 min. Intracellular killing of the target organisms was evaluated by determining the number of surviving yeast cells after co-incubation of C. albicans and human whole blood. EPs[R] 7630 was applied in therapeutically relevant concentrations between 0 and 30 [micro]g/ml.

Compared with controls EPs[R] 7630 increased the number of phagocytosing PBP during the observed time points between 2 and 10 min in a concentration-dependent manner, with a maximum enhancement of 56% at 2 min (p = 0.002). The application of EPs[R] 7630 also led to a significant increase in the number of burst-active PBP for all time points observed beyond 2 min (p <0.001). The maximum augmentation was 120% after application of 30 [micro]g/ml EPs[R] 7630 at 4 min. Using a microbiological assay, intracellular killing was also enhanced by EPs[R] 7630. This was expressed by a significant reduction in the number of surviving target organisms (p < 0.001). The maximum reduction in viable yeast cells (-31%) was observed after co-incubation for 120 min with the highest concentration of EPs[R] 7630 (30 [micro]g/ml).

In conclusion, the positive effects of EPs[R] 7630 on phagocytosis, oxidative burst, and intracellular killing of yeast cells as test organisms are important components of the compound's biological activity. Our findings constitute a valuable contribution to understanding the clinical effects of EPs[R] 7630.

[c] 2006 Elsevier GmbH. All rights reserved.

Keywords: Pelargonium sidoides; EPs[R] 7630; Phagocytosis; Oxidative burst; Intracellular killing; Flow cytometry

Introduction

The anti-infective properties of the roots of some Geraniaceae-species such as Pelargonium sidoides are well-known in traditional South African tribal medicine. Today, a commercialized extract derived from the roots of Pelargonium sidoides, termed EPs[R] 7630, is available for treatment of upper respiratory tract infections (URTI). Recent randomized placebo-controlled trials have demonstrated that EPs[R] 7630 is superior to placebo in the therapy of acute bronchitis and tonsillopharyngitis (Matthys et al., 2003; Bereznoy et al., 2003). In order to explain its clinical activity, antiviral, antibacterial, immunomodulating, and secretolytic properties have been suggested as the preparation's essential modes of action.

Previous in vitro-investigations have revealed immunomodulating effects by induction of TNF[alpha], interferons, and NO. Furthermore, a significant influence on the ciliary system of the respiratory tract as well as some weak antibacterial properties against various gram-positive and gram-negative bacteria have been described (Kolodziej et al., 2003; Kayser et al., 2001; Kayser and Kolodziej, 1997; Neugebauer et al., 2005).

However, the detailed pharmacological effects of EPs[R] 7630 are not clear and still need to be determined. Therefore, the in vitro-study presented here aimed to characterize the impact of EPs[R] 7630 on the phagocytic activity of human peripheral blood phagocytes (PBP). Phagocytosis and related PBP activity play a pivotal role in the innate immunity of host defence against invading pathogens such as bacteria, viruses or fungi (Henneke and Golenbock, 2004; Salih et al., 2000). Circulating PBP are rapidly recruited to sites of infection by host-and/or pathogen-derived components, which also prime these host cells for enhanced microbicidal activity (Kobayashi et al., 2005). After binding and ingesting the microorganisms (phagocytosis) by the PBP, the production and release of reactive oxygen species (oxidative burst) is triggered, which contributes to the microbial death pathway, eventually leading to the elimination of the pathogen (intracellular killing) (Greenberg and Grinstein, 2002). Thus, enhanced PBP activity under the influence of EPs[R] 7630 may contribute to the anti-infective properties of the preparation. In order to draw a complete picture of the PBP activity, in this study the impact of EPs[R] 7630 on phagocytosis, oxidative burst, and intracellular killing was evaluated.

Materials and methods

Target organism and fluorescence labelling

Candida albicans DSM 1386 (ATCC 10231) was used as target organism and cultured in tryptic soy broth (TSB; Oxoid, UK). The number of yeast cells was assessed by microscope with a Neubauer's counting chamber. C. albicans was labelled with the green fluorescent marker calcein-acetoxymethylester (calcein-AM, Molecular Probes/Invitrogen, Germany) a membrane permeable ester which is intracellularly converted into a non-permeable acid by esterases. The yeast cells were washed three times in Dulbecco's phosphate-buffered saline (PBS; Sigma, Germany) and then incubated in PBS containing 1% glucose and 6.25ng/ml calcein-AM at 37 [degrees]C for 50 min in a thermomixer (Eppendorf, Germany) at 1100 rpm. The cells were washed three times in PBS to remove excess calcein-AM, and then resuspended in RPMI-1640 (Sigma, Germany). Labelled yeast organisms were stored at 4 [degrees]C for a maximum of 7 days.

Test substance

EPs[R] 7630 is a proprietary liquid herbal drug preparation of the root of Pelargonium sidoides (drug/extract ratio of 1:8-10) using aqueous ethanol (11% (m/m)) as extraction solvent. The active ingredients of EPs[R] 7630 include highly oxygenated coumarins (e.g. umckalin) and polyphenolic compounds. A single batch (PSC343/Ch001) of dried EPs[R] 7630 (Spitzner/Schwabe, Ettlingen/Karlsruhe, Germany) was used for all the experiments. For application in the in vitro test systems EPs[R] 7630 was dissolved in sterile water in order to prepare a stock solution (1000 [micro]g/ml). For the phagocytosis and killing assays the stock solution was directly added to the samples in order to achieve the final concentrations of 3, 10, and 30 [micro]g/ml EPs[R] 7630, which represent therapeutically relevant concentrations. Blood samples were pre-treated with EPs[R] 7630 (see below). In order to maintain the desired concentrations of the test substance during co-incubation EPs[R] 7630 was also added to the yeast cells directly before performing the phagocytosis/burst and killing assays.

Blood samples and pre-treatment of blood samples

Ten millilitres of blood were collected from healthy volunteers who had no acute or chronic disease and were not taking any regular medication. Anticoagulation was achieved by adding 5 IE heparin/ml blood (Liquemin N 5000, Roche, Germany). The concentration of PBP was determined by microscope with a Neubauer's counting chamber. Blood samples were pretreated with EPs[R] 7630 at 37 [degrees]C for 30 min in a Certomat[R] H/U (B. Braun, Germany). Samples for the analysis of oxidative burst were additionally incubated with 0.5 [micro]g/ml dihydroethidium (DHE; Sigma, Germany) for the last 10 min of pre-treatment. DHE allows investigation of the burst reaction since reactive oxygen species oxidize the dye, thus converting it from colourless to red fluorescent. All pre-treated blood samples were cooled on ice for 25 min.

Evaluation of phagocytosis and oxidative burst

In order to determine phagocytosis and oxidative burst of human PBP, a flow cytometry-based, whole blood approach was used as described previously (Salih et al., 2000; Anding et al., 2003; Frank et al., 2003). Measurement of phagocytosis and oxidative burst was performed together in one test tube. Labelled yeast cells were mixed with the anti-coagulated and pre-treated blood in a ratio of one target organism to one PBP. Aliquots were then agitated in a thermomixer at 37 [degrees]C and 1100 rpm. Co-incubation was stopped after 0, 2, 4, 6, 10, and 30 min by adding 400 [micro]l of a 100 mM N-ethyl-maleinimide-solution (NEM; Sigma, Germany), and the samples were placed on ice to avoid further phagocytosis and burst. The test tubes were then washed at 250g and 4 [degrees]C for 5 min, and the PBP were stained with orange fluorescent anti-CD13-RPE antibodies (Dako, Denmark). For labelling, the pellet was re-suspended with 100 [micro]l PBS containing 1.8 [micro]l of the antibody solution and incubated at room temperature for 20 min. Next, the erythrocytes were lysed by a 10-min incubation with ice-cold lysis buffer containing ammonium chloride (Sigma, Germany), potassium hydrogen carbonate, and sodium-EDTA (Sigma, Germany). After one additional washing (250g, 4 [degrees]C, 5 min), the samples were stored at 4 [degrees]C and measured within 2 h.

Flow cytometry

Samples were measured with a FACScan flow cytometer (Becton Dickinson, Germany). Data were collected using linear amplifiers for the forward scatter (FSC), and logarithmic amplifiers for the side scatter (SSC), FL1, FL2, and FL3. Prior to analysis samples containing blood with or without yeast cells as well as all variations of red, orange, and green fluorescence served as controls. For assessment of phagocytosis/burst 2500 PBP were counted per assay. The flow cytometry data were analyzed using integrated Lysis II software. The assay allows reading out for the percentage of phagocytosing PBP and the percentage of PBP with burst reaction after phagocytosis.

Yeast cell killing assay

For assessment of intracellular killing, PBP blood samples were pre-treated with EPs[R] 7630 as explained before. However, nonlabeled yeast cells were used in this assay and the ratio of target cells to PBP was also adjusted to 1:1. Co-incubation of blood and target organisms was performed in a thermomixer (see above) and stopped after 0, 15, 30, 60, and 120 min by adding 400 [micro]l of a lysis solution comprising Triton X (Sigma, Germany) and Tween 20 (Sigma, Germany). During subsequent incubation in a thermomixer at 37 [degrees]C and 1100 rpm, all blood cells were lysed, while the yeast cells were kept unlysed. The samples were then centrifuged at 1000g and 4 [degrees]C for 5 min. The pellet was resuspended in PBS. In order to determine the intracellular killing of the target organisms serial dilutions of the suspension were plated on DST-agar-plates and incubated at 37 [degrees]C. Colony forming units (cfu) were counted after 24 and 48 h.

Statistical analysis

Data were analyzed with SPSS[R] for Windows[R] V11.5. A repeated measures analysis of variance (ANOVA) was performed. P-values were corrected for sphericity according to Huynh-Feldt (Huynh and Feldt, 1976). All P-values stated refer to factor "concentration" of the test substance applied.

Results

Enhanced phagocytosis by EPs[R] 7630

The flow cytometric investigations revealed that EPs[R] 7630 improves the velocity with which PBP are recruited for phagocytosis in a concentration-dependent manner (Fig. 1A). In control samples without the test substance the percentage of phagocytosing PBP increased over time to just under 50% after 30 min. After application of the test substance the number of phagocytosing PBP was augmented for the observed time points between 2 and 10 min (p = 0.002, repeated measures ANOVA for all time points; factor "concentration"). When 30 [micro]g/ml EPs[R] 7630 were added, compared to the controls the number of phagocytosing PBP was enhanced by 56% after 2min, by 54% after 4min, by 45% after 6 min, and by 15% after 10 min. However, since the process of ingesting the target organisms requires some time, no effect by the test substance could be observed at the start (0 min) of the assay. Control experiments showed that EPs[R] 7630 had no significant impact on adhesion of target cells to the surface of PBP (data not shown).

Improved oxidative burst by EPs[R] 7630

When challenged with the target organisms the burst response by the PBP also improved significantly under the influence of EPs[R] 7630 (Fig. 1B; p<0.001, repeated measures ANOVA; factor "concentration"). While the number of PBP that released reactive oxygen compounds in the control samples increased from under 10% at the start of co-incubation (0 min) to over 60% after 30 min, the number of burst-active PBP increased in a concentration-dependent manner by the addition of the test substance. The application of 30 [micro]g/ml EPs[R] 7630 led to an 89% increase of burst-active PBP at the start of co-incubation (0 min), to 86% at 2 min, 120% at 4 min, 81% at 6 min, 37% at 10 min, and was still 20% at 30 min. Thus, under the influence of EPs[R] 7630 the PBP are able to release the burst substances either more quickly or more easily, so that in the course of an infection more PBP are able to fight the pathogens earlier by burst production.

[FIGURE 1 OMITTED]

Increased intracellular killing by EPs[R] 7630

Originally, it was planned to evaluate intracellular killing using a flow cytometric technique based on the detection of dead yeast cells by labelling with ethidium homodimer-1 (EthD-1). However, control experiments using heat killed C. albicans revealed that EPs[R] 7630 concentration-dependently interfered with the DNA dye EthD-1 resulting in reduced fluorescence signals. In contrast, control experiments with calcein-AM stained yeast cells and both DHE pre-treated and anti-CD13 stained PBP had proven that EPs[R] 7630 did not interfere with the dyes used for assessment of phagocytosis and burst.

[FIGURE 2 OMITTED]

Because of the methodical limitations of flow cytometric evaluation of intracellular killing in the presence of the test substance, a microbiological killing assay was used as an alternative approach. Independently of the test substance, 76% of the target cell inoculum was eliminated by intracellular killing within 120 min in the control samples. While EPs[R] 7630 had no effect on intracellular killing at a concentration of 10 [micro]g/ml, in the presence of 30 [micro]g/ml of EPs[R] 7630 the number of surviving test organisms was reduced by on average 15% at 15 min, 25% at 3 min, 27% at 60 min and by 31% at 120 min (Fig. 2; p<0.001 repeated measures ANOVA, factor "concentration"). Examination of the impact of EPs[R] 7630 on the growth and survival of C. albicans verified that there was no direct antifungal activity at the concentrations of EPs[R] 7630 used on the target organisms. From this we conclude that the improved intracellular killing is caused by a positive effect of the extract on the PBP killing activity.

Discussion

Phagocytosis and associated PBP activity are crucial effector mechanisms both in the first line of the host defence against invading pathogens and also to resolve the infection once established. Our investigations demonstrate clearly that the Pelargonium-derived special extract EPs[R] 7630 significantly improves PBP function.

The increased number of phagocytosing PBP under the influence of EPs[R] 7630 shows an improved velocity of PBP recruitment. The enhanced phagocytic capacity was also correlated with an increased number of phagocytosed target organisms (data not shown). The observation that the stimulating effect of EPs[R] 7630 on the phagocytic capacity of PBP seems to increase no further after 30 min of co-incubation is due to the limited number of free target organisms within the experimental setting. Since the target to effector cell ratio was adjusted to 1:1, all yeast cells were ingested after 30 min. However, the stimulation of the burst activity is still visible at 30 min and is thus still detectable at that point even when all test organisms have already been ingested. This indicates that EPs[R] 7630 sustainably improves PBP activity.

While some authors narrow their investigations of phagocytosis to evaluation of target organisms or target particles with or without determination of oxidative burst, intracellular killing is an essential additional aspect of phagocytosis and should also be investigated (Anding et al., 2003). Our investigations additionally revealed that EPs[R] 7630 significantly improves intracellular killing. Therefore, EPs[R] 7630 not only enhances the phagocytic capacity and oxidative burst, but eventually leads to efficient elimination of the invading pathogens. Because PBP activity not only represents an innate immune effector but also a bridge between the innate and acquired immune responses (Greenberg and Grinstein, 2002) EPs[R] 7630 provides significant immunomodulating properties. Improved activities of various PBP functions under the influence of EPs[R] 7630 protect from URTI of various aetiologies and accelerate convalescence once an infection has become established. EPs[R] 7630 is a liquid formulation which is administered orally. In the course of an URTI the preparation is therefore able to act locally on both the resident and invading phagocytes at the site of inflammation.

The herbal drug preparation EPs[R] 7630 has been used for all the clinical trials performed so far. To emulate as far as possible the clinical setting, we decided to use therapeutically relevant concentrations of the extract. However, like many other phytotherapeutic preparations, the extract is regarded as the active ingredient consisting of various pharmacologically active substances which also explains why these types of phytotherapeutic extracts may display diverse modes of action. Amongst others EPs[R] 7630 contains highly oxygenated coumarins (e.g. umckalin) and polyphenolic compounds. However, it is not clear which of the active ingredients accounts for the observed effects on leucocyte activity. Further investigations are already under way addressing these questions.

In summary, the positive effects of EPs[R] 7630 on phagocytosis, oxidative burst, and intracellular killing of yeast cells as test organisms are important components of the compound's biological activity. Our findings constitute a valuable contribution towards understanding its clinical effects.

Acknowledgements

The authors thank Deborah Lawrie-Blum for reviewing the manuscript. This work was funded by a grant from Spitzner Pharmaceuticals, Ettlingen, Germany.

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Andreas Conrad*, Cathrin Hansmann, Inge Engels, Franz D. Daschner, Uwe Frank

Institute of Environmental Medicine and Hospital Epidemiology, University Medical Center Freiburg, Breisacher Strasse 115B, 79106, Freiburg, Germany

*Corresponding author. Tel.: +49 761 270 8262; fax: +49 761 270 8253.

E-mail address: andreas.conreas.corad@uniklinik-freiburg.de (A. Conrad).
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Author:Conrad, Andreas; Hansmann, Cathrin; Engels, Inge; Daschner, Franz D.; Frank, Uwe
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:4EUGE
Date:Feb 1, 2007
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