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EPs® 7630, an extract from Pelargonium sidoides roots inhibits adherence of Helicobacter pylori to gastric epithelial cells
ARTICLE IN PRESS
Phytomedicine 14 (2007) SVI 5–8 www.elsevier.de/phymed
EPss 7630, an extract from Pelargonium sidoides roots inhibits adherence of Helicobacter pylori to gastric epithelial cells W. Beil, P. Kilian Department of Pharmacology, Hannover Medical University, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany
Abstract Helicobacter pylori specifically adheres to gastric host cells, mainly based on carbohydrate-mediated cell–cell interaction. The extract of Pelargonium sidoides roots (EPss 7630), a South African herbal remedy, is currently used to treat acute bronchitis. EPss 7630 prevents bacteria from attaching to cell membranes. Therefore, the ability of EPss 7630 to interfere with H. pylori growth and adhesion to gastric epithelial cells (AGS cells) was tested in vitro. EPss 7630 inhibited H. pylori growth and with higher potency adhesion to gastric AGS cells. EPss 7630 (50 and 100 mg/ml) reduced bacterial count attached to AGS cells by 77% and 91%, respectively. The results suggest that the mode of action of EPss 7630 is mainly related to its anti-adhesive activity. r 2006 Elsevier GmbH. All rights reserved. Keywords: Pelargonium sidoides; Helicobacter pylori; Adhesion; Gastric epithelial cells
Introduction Helicobacter pylori is a Gram-negative spiral bacillus that was isolated from the stomach of a patient with chronic active gastritis (Marshall and Warren, 1983). H. pylori is considered to be an important factor in peptic ulcers and there is evidence that the risk of gastric cancer is increased by infection with the bacterium. Moreover, cure of H. pylori infection has been associated with regression of gastric MALT lymphoma (van Amsterdam et al., 2006). The first line regimen for eradication of H. pylori infection currently includes a triple therapy, which combines two antibiotics (amoxicillin plus clarithromycin or metronidazole) and a proton pump inhibitor. Corresponding author. Tel.: +49 511 5322951; fax: +49 511 5322794. E-mail address: [email protected] (W. Beil).
0944-7113/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2006.11.024
With this regimens eradication rates of 70–90% are obtained (van der Hulst et al., 1996). It is accepted that antibiotic resistance is the main factor affecting outcome of standard treatments for H. pylori eradication (Perri et al., 2001). A large USA survey has shown an overall prevalence of resistance to clarithromycin, metronidazole and amoxicillin of 10.1%, 36.9% and 1.4%, respectively (Meyer et al., 2002). Several herbal medicines have been tested for antibacterial activity against H. pylori in vitro and in clinical studies as possible candidates for use in modified eradication therapy (Li et al., 2005; Tominaga et al., 2005; Takashima et al., 2002; Higuchi et al., 1999). An extract of Pelargonium sidoides (EPss 7630), a South African herbal remedy, is currently used to treat acute bronchitis (Brown, 2004). In vitro studies suggest that the extract has antimicrobial and immunomodulatory properties (Kayser and Kolodziej, 1997; Kolodziej et al., 2003).
ARTICLE IN PRESS 6
W. Beil, P. Kilian / Phytomedicine 14 (2007) SVI 5–8
The aim of the present study was to examine the antibacterial activity of EPss 7630 against H. pylori in vitro in coculture with gastric epithelial cells.
Methods H. pylori strains and culture conditions Three clinical isolates of H. pylori were used: Hp 4 derived from a patient with duodenal ulcer, Hp 39 derived from a patient with chronic gastritis and Hp 94 derived from a patient with gastric ulcer. All isolates were cag-A-negative and noncytotoxin producing strains. Presence of cagA was analysed by RT-PCR as described (Wagner et al., 1997). Presence of vacuolating toxin was determined by incubation of gastric HM02 cells with concentrated supernatants from H. pylori broth medium. H. pylori was grown on Columbia agar for 48 h under microaerophilic conditions. Bacteria were harvested in RPMI 1640, washed twice and centrifugated at 11,000g for 10 min. For estimation of the desired bacterial number, bacteria were resuspended in RPMI 1640, optical density measurements were performed at 600 nm and were correlated to viable colony forming counts (cfu).
3 h cells were washed to remove non-adherent bacteria and test compounds (EPss 7630 1–100 mg/ml or amoxicillin 8–64 mg/ml) were added. After 21 h incubation H. pylori density was determined by urease test. Urease test medium was: 0.9% NaCl, 1 mM citric acid titrated with 1 N NaOH to pH 7.4, urea 20 mM and 14 mg/ml phenol red. The colormetric changes, proportional to urease activity, were determined by spectrophotometric analysis at an optical density of 555 nm.
Testing for adherence of H. pylori to gastric epithelial cells H. pylori strains were resuspended (OD: 2.0) in 1 ml RPMI and were incubated with 10 ml 0.1% FITC in DMSO for 1 h at room temperature in the dark. The FITC-labelled bacteria were then washed three times in RPMI containing 0.1% Tween 20. AGS monolayers were infected with FITC-labelled bacteria (5 106 cfu) in the absence and presence of EPss 7630 (50 and 100 mg/ml). The cell monolayers were washed after 3 h in RPMI, lysed at 37 1C in trypsin-EDTA and bacterial adherence was quantified by fluorescence (FITC excited at 485 nm and detected at 528 nm).
Statistics Testing for antibacterial activity 7
Results are expressed as mean7SE of at least three experiments with each H. pylori strain. For statistical analysis Student’s t-test was used. Probability values o0.05 were considered significant.
H. pylori (1.5 10 cfu, OD ¼ 0.1) were inoculated into 1 ml brain heart infusion (BHI) broth supplemented with 10% heat-inactivated foetal calf serum (FCS) containing 1–100 mg of an extract of Pelargonium sidoides roots, referred to as EPss 7630 (from Dr. Willmar Schwabe Pharmaceuticals, Karlsruhe, Germany). The extract was dissolved in 12% ethanol and sterilised with a 0.2 mm filter. After 48 h of microaerophilic incubation at 37 1C the turbidity of the medium was monitored at 600 nm. Antibacterial activity of amoxicillin (Sigma, Munich, Germany) was tested by E test. Columbia Agar plates were inoculated with 108 H. pylori by using a sterile swab to cover the plate surface. E test strips were added and the plates were incubated for 48 h under microaerophilic conditions.
During two days incubation in BHI broth the H. pylori count increased from 1 107 to 2.970.04 107 cfu. EPss 7630 (1–50 mg/ml) did not inhibit H. pylori growth. At 100 mg/ml H. pylori growth was reduced to 43710% of control value (po0.05).
Testing for antibacterial activity in coculture with AGS cells
Effect of EPss 7630 and amoxicillin on H. pylori count adherent to AGS cells
The gastric cancer cell line AGS (ATCC CRL 1739) was plated at a density of 104 cells in 96-well-plates in 0.2 ml RPMI 1640 with 10% FBS at 37 1C in a humified atmosphere of 5% CO2 in air and left to adhere for 24 h. Subsequently, cell monolayers were washed with RPMI 1640 and were infected with 105 H. pylori in 0.2 ml medium (70% RPMI 1640, 20% BHI, 10% FCS). After
During the 3 h infection period approximately 30% of the bacteria (3 104 cfu) attached to AGS cells. During the following 21 h incubation period H. pylori density increased to 6–7 105 cfu, i.e. the bacteria replicate. The effects of EPss 7630 and amoxicillin on H. pylori density are shown in Figs. 1 and 2. EPss 7630 (50 and 100 mg/ml) significantly reduced H. pylori density at
Results Inhibition of H. pylori growth by EPss 7630 in BHI medium
ARTICLE IN PRESS W. Beil, P. Kilian / Phytomedicine 14 (2007) SVI 5–8
100
60 *
*
0 0
5
10 50 EPs® 7630 (µg/ml)
100
Fig. 1. Effect of EPss 7630 on H. pylori density attached to AGS cells. AGS cell monolayers were infected with 105 H. pylori. After 3 h cell monolayers were washed to remove nonadherent bacteria and EPss 7630 was added. After 21 h incubation H. pylori density was determined by urease test. Data are expressed as percentage of control (mean7SD of three experiments with Hp 4, 39 and 94). *po0.05 vs. control.
*
*
50
0
0
50 EPs® 7630 (µg/ml)
100
Fig. 3. Effect of EPss 7630 on H. pylori adherence to AGS cells. AGS cell monolayers were infected with 5 106 FITClabelled H. pylori in the presence of EPss 7630. Cell monolayer were washed after 3 h to remove non-adherent bacteria and bacterial adherence was quantified by fluorescence. Data are expressed as percentage of control (mean7SD of three experiments with Hp 4, 39 and 94). *po0.05 vs. control.
EPss 7630 (50 and 100 mg/ml) significantly reduced H. pylori adherence to AGS cells.
100 % H. pylori count
(%) H. pylori count
% H. pylori count
120
7
Discussion
50
0 0
8
16 32 Amoxicillin (µg/ml)
64
Fig. 2. Effect of amoxicillin on H. pylori density attached to AGS cells. AGS cell monolayers were infected with 105 H. pylori. After 3 h cell monolayers were washed to remove nonadherent bacteria and amoxicillin was added. After 21 h incubation H. pylori density was determined by urease test. Data are expressed as percentage of control (mean7SD of three experiments with Hp 4, 39 and 94).
AGS cells to 2375% and 18.775% of control value, respectively. Amoxicillin failed to block H. pylori growth. It should be noted that all H. pylori strains used were susceptible to amoxicillin as determined by E test (data not shown).
EPss 7630 reduces H. pylori adherence to AGS cells To investigate whether EPss 7630 inhibits the adhesion of H. pylori to AGS cells, an in vitro binding assay was used. FITC-labelled H. pylori were overlaid onto AGS cell monolayers in the absence and presence of EPss 7630. After three hours the cell monolayers were washed and the extent of bacteria adhesion was quantified by fluorometric analysis. As shown in Fig. 3
In the present study, we demonstrate that EPss 7630 inhibits (i) H. pylori growth and (ii) reduces bacterial adherence to gastric epithelial AGS cells. A suppressive effect of EPss 7630 against H. pylori growth in BHI broth was observed at a concentration of 100 mg/ml. A significant reduction of H. pylori adherence was observed with EPss 7630 at concentrations X50 mg/ml (Fig. 3). EPss 7630 (50 and 100 mg/ml) reduced density of attached H. pylori during a 21 h incubation period by 77% and 91% (Fig. 2). We, therefore, suggest that the mode of action of EPss 7630 is mainly related to its antiadhesion activity. Bacterial adherence to mucosal surfaces is generally considered to be an important prerequisite for colonisation and infection. H. pylori shows a wide spectrum of different specificities in adhesion to host cells. Several surface carbohydrates mediating cell adhesions have been identified, such as the blood group antigen-binding adhesion (BabA) which recognise the Leb antigen, the sialyl-dimeric-Lewisx glycosphingolid, the sialic acidbinding adhesin (SabA) and sulphated glycosaminoglycans such as heparan sulphate (Lamarque and Peek, 2003; Ruiz-Bustos et al., 2001). It has been reported that sialyllactose, an inhibitor of SabA, significantly reduced the load of the bacteria to gastrointestinal epithelial cells (Simon et al., 1997). Moreover, it was shown that acidic polysaccharides rich of uronic acid from Artemisia capillaris and Panax ginseng inhibited H. pylori adhesion to gastric epithelial cells (Lee et al., 2004).
ARTICLE IN PRESS 8
W. Beil, P. Kilian / Phytomedicine 14 (2007) SVI 5–8
The bioactive ingredients in Pelargonium sidoides are the tri- and tetra-oxygenated coumarins, gallic acid, gallic acid methyl esters and various flavonoids. Pelargonium sidoides contains two distinct coumarins: umckalin and three unique coumarin sulphates (Latte´ et al., 2000). In the present study, we did not test the effect of isolated constituents of the extract on H. pylori adherence. However, it was postulated that negatively charged groups may play a role in host cell–H. pylori adhesion (Lee et al., 2004). Heparan sulphate binding to H. pylori was inhibited by sulphated polysaccharides (heparin and chondroitin sulphates) (Hirmo et al., 1995). Therefore, we suggest that negatively charged components of the extract bind to H. pylori and prevent binding of the bacteria to AGS cells. The antibiotic amoxicillin failed to affect H. pylori growth in co-culture with AGS cells. These results indicate that when H. pylori binds to the AGS cells, the susceptibility to an antibiotic is altered. The concentrations of amoxicillin used were multiples of the MIC values, determined by E test. The mechanism of this resistance is not known. AGS cells are mucous producing cells and form a thin mucous layer which can modify the antimicrobial activity (the biofilm phenomenon). This phenomenon has been shown with H. pylori attached to Hep-2 cells (Me´graud et al., 1991). We conclude that EPss 7630 reduces H. pylori density in vitro by blocking adhesion of H. pylori to gastric host cells.
References Brown, D.J., 2004. Extract of Pelargonium sidoides: South African herbal remedy successfully treats acute bronchitis and tonsillopharyngitis. Herbal Gram. 63, 17–19. Higuchi, K., Arakawa, T., Ando, K., Fujiwara, Y., Uchida, T., Kuroki, T., 1999. Eradication of Helicobacter pylori with a Chinese herbal medicine without emergence of resistant colonies. Am. J. Gastroenterol. 94, 1419–1420. Hirmo, S., Utt, M., Ringner, M., Wadstro¨m, T., 1995. Inhibition of heparan sulphate and other glycosaminoglycans binding to Helicobacter pylori by various polysulphated carbohydrates. FEMS Immunol. Med. Microbiol. 10 (3–4), 301–306. Kayser, O., Kolodziej, H., 1997. Antibacterial activity of extracts and constituents of Pelargonium sidoides and Pelargonium reniforme. Planta Med. 63 (6), 508–510. Kolodziej, H., Kayser, O., Radtke, O.A., Kiderlen, A.F., Koch, E., 2003. Pharmacological profile of extracts of Pelargonium sidoides and their constituents. Phytomedicine 10 (Suppl. 4), 18–24. Lamarque, D., Peek Jr., R.M., 2003. Pathogenesis of Helicobacter pylori infection. Helicobacter 8 (Suppl. 1), 21–30. Latte´, K.P., Kayser, O., Tan, N., Kaloga, M., Kolodziej, H., 2000. Unusual coumarin patterns of Pelargonium species
forming the origin of the traditional herbal medicine umckaloabo. Z. Naturfosch. 55c, 528–533. Lee, J.H., Park, E.K., Uhm, C.-S., Chung, M.-S., Kim, K.H., 2004. Inhibition of Helicobacter pylori adhesion to human gastric adenocarcinoma epithelial cells by acidic polysaccharides from Artemisia capillaris and Panax ginseng. Planta Med. 70, 615–619. Li, Y., Xu, C., Zhang, Q., Liu, J.Y., Tan, R.X., 2005. In vitro anti-Helicobacter pylori action of 30 Chinese herbal medicines used to treat ulcer diseases. J. Ethnopharmacol. 98 (3), 329–333. Marshall, B., Warren, J.R., 1983. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet i, 1273–1275. Me´graud, F., Trimoulet, P., Lamouliatte, H., Boyanova, L., 1991. Bactericidal effect of amoxicillin on Helicobacter pylori in an in vitro model using epithelial cells. Antimicrob. Agents Chemother. 35 (5), 869–872. Meyer, J.M., Silliman, N.P., Wang, W., Siepman, N.Y., Sugg, J.E., Morris, D., Zhang, J., Bhattacharyya, H., King, E.C., Hopkins, R.J., 2002. Risk factors for Helicobacter pylori resistance in the United States: the surveillance of H. pylori antimicrobial resistance partnership (SHARP) study, 1993–1999. Ann. Intern. Med. 136 (1), 13–24. Perri, F., Villani, M.R., Festa, V., Quitadamo, M., Andriulli, A., 2001. Predictors of failure of Helicobacter pylori eradication with the standard ‘Maastricht triple therapy’. Aliment. Pharmacol. Ther. 15 (7), 1023–1029. Ruiz-Bustos, E., Ochoa, J.L., Wadstro¨m, T., Ascencio, F., 2001. Isolation and characterisation of putative adhesins from Helicobacter pylori with affinity for heparan sulphate proteoglycan. J. Med. Microbiol. 50, 215–222. Simon, P.M., Goode, P.L., Mobasseri, A., Zopf, D., 1997. Inhibition of Helicobacter pylori binding to gastrointestinal epithelial cells by sialic acid-containing oligosaccharides. Infect. Immun. 65 (2), 750–757. Takashima, J., Chiba, N., Yoneda, K., Ohsaki, A., 2002. Derrisin, a new rotenoid from Derris malaccensis plain and anti-Helicobacter pylori activity of its related constituents. J. Nat. Prod. 65, 611–613. Tominaga, K., Higuchi, K., Hamasaki, N., Tanigawa, T., Sasaki, E., Watanabe, T., Fujiwara, Y., Oshitani, N., Arakawa, T., Ishii, E., Tezuka, Y., Nagaoka, T., Kadota, S., 2005. Antibacterial activity of a Chinese herbal medicine, Gosyuyu (Wu–Chu–Yu) against Helicobacter pylori. Nippon Rinsho 63 (Suppl. 11), 592–599. van Amsterdam, K., van Vliet, A.H., Kusters, J.G., van der Ende, A., 2006. Of microbe and man: determinants of Helicobacter pylori-related diseases. FEMS Microbiol. Rev. 30 (1), 131–156. van der Hulst, R., Keller, J.J., Rauwas, E.A., Tytgat, G.N., 1996. Treatment of Helicobacter pylori infection: a review of the world literature. Helicobacter 1, 6–19. Wagner, S., Beil., W., Westermann, J., Logan, R.P., Bock, C.T., Trautwein, C., Bleck, J.S., Manns, M.P., 1997. Regulation of gastric epithelial cell growth by Helicobacter pylori: evidence for a major role of apoptosis. Gastroenterology 113, 1836–1847.
Phytomedicine 14 (2007) SVI 5–8 www.elsevier.de/phymed
EPss 7630, an extract from Pelargonium sidoides roots inhibits adherence of Helicobacter pylori to gastric epithelial cells W. Beil, P. Kilian Department of Pharmacology, Hannover Medical University, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany
Abstract Helicobacter pylori specifically adheres to gastric host cells, mainly based on carbohydrate-mediated cell–cell interaction. The extract of Pelargonium sidoides roots (EPss 7630), a South African herbal remedy, is currently used to treat acute bronchitis. EPss 7630 prevents bacteria from attaching to cell membranes. Therefore, the ability of EPss 7630 to interfere with H. pylori growth and adhesion to gastric epithelial cells (AGS cells) was tested in vitro. EPss 7630 inhibited H. pylori growth and with higher potency adhesion to gastric AGS cells. EPss 7630 (50 and 100 mg/ml) reduced bacterial count attached to AGS cells by 77% and 91%, respectively. The results suggest that the mode of action of EPss 7630 is mainly related to its anti-adhesive activity. r 2006 Elsevier GmbH. All rights reserved. Keywords: Pelargonium sidoides; Helicobacter pylori; Adhesion; Gastric epithelial cells
Introduction Helicobacter pylori is a Gram-negative spiral bacillus that was isolated from the stomach of a patient with chronic active gastritis (Marshall and Warren, 1983). H. pylori is considered to be an important factor in peptic ulcers and there is evidence that the risk of gastric cancer is increased by infection with the bacterium. Moreover, cure of H. pylori infection has been associated with regression of gastric MALT lymphoma (van Amsterdam et al., 2006). The first line regimen for eradication of H. pylori infection currently includes a triple therapy, which combines two antibiotics (amoxicillin plus clarithromycin or metronidazole) and a proton pump inhibitor. Corresponding author. Tel.: +49 511 5322951; fax: +49 511 5322794. E-mail address: [email protected] (W. Beil).
0944-7113/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2006.11.024
With this regimens eradication rates of 70–90% are obtained (van der Hulst et al., 1996). It is accepted that antibiotic resistance is the main factor affecting outcome of standard treatments for H. pylori eradication (Perri et al., 2001). A large USA survey has shown an overall prevalence of resistance to clarithromycin, metronidazole and amoxicillin of 10.1%, 36.9% and 1.4%, respectively (Meyer et al., 2002). Several herbal medicines have been tested for antibacterial activity against H. pylori in vitro and in clinical studies as possible candidates for use in modified eradication therapy (Li et al., 2005; Tominaga et al., 2005; Takashima et al., 2002; Higuchi et al., 1999). An extract of Pelargonium sidoides (EPss 7630), a South African herbal remedy, is currently used to treat acute bronchitis (Brown, 2004). In vitro studies suggest that the extract has antimicrobial and immunomodulatory properties (Kayser and Kolodziej, 1997; Kolodziej et al., 2003).
ARTICLE IN PRESS 6
W. Beil, P. Kilian / Phytomedicine 14 (2007) SVI 5–8
The aim of the present study was to examine the antibacterial activity of EPss 7630 against H. pylori in vitro in coculture with gastric epithelial cells.
Methods H. pylori strains and culture conditions Three clinical isolates of H. pylori were used: Hp 4 derived from a patient with duodenal ulcer, Hp 39 derived from a patient with chronic gastritis and Hp 94 derived from a patient with gastric ulcer. All isolates were cag-A-negative and noncytotoxin producing strains. Presence of cagA was analysed by RT-PCR as described (Wagner et al., 1997). Presence of vacuolating toxin was determined by incubation of gastric HM02 cells with concentrated supernatants from H. pylori broth medium. H. pylori was grown on Columbia agar for 48 h under microaerophilic conditions. Bacteria were harvested in RPMI 1640, washed twice and centrifugated at 11,000g for 10 min. For estimation of the desired bacterial number, bacteria were resuspended in RPMI 1640, optical density measurements were performed at 600 nm and were correlated to viable colony forming counts (cfu).
3 h cells were washed to remove non-adherent bacteria and test compounds (EPss 7630 1–100 mg/ml or amoxicillin 8–64 mg/ml) were added. After 21 h incubation H. pylori density was determined by urease test. Urease test medium was: 0.9% NaCl, 1 mM citric acid titrated with 1 N NaOH to pH 7.4, urea 20 mM and 14 mg/ml phenol red. The colormetric changes, proportional to urease activity, were determined by spectrophotometric analysis at an optical density of 555 nm.
Testing for adherence of H. pylori to gastric epithelial cells H. pylori strains were resuspended (OD: 2.0) in 1 ml RPMI and were incubated with 10 ml 0.1% FITC in DMSO for 1 h at room temperature in the dark. The FITC-labelled bacteria were then washed three times in RPMI containing 0.1% Tween 20. AGS monolayers were infected with FITC-labelled bacteria (5 106 cfu) in the absence and presence of EPss 7630 (50 and 100 mg/ml). The cell monolayers were washed after 3 h in RPMI, lysed at 37 1C in trypsin-EDTA and bacterial adherence was quantified by fluorescence (FITC excited at 485 nm and detected at 528 nm).
Statistics Testing for antibacterial activity 7
Results are expressed as mean7SE of at least three experiments with each H. pylori strain. For statistical analysis Student’s t-test was used. Probability values o0.05 were considered significant.
H. pylori (1.5 10 cfu, OD ¼ 0.1) were inoculated into 1 ml brain heart infusion (BHI) broth supplemented with 10% heat-inactivated foetal calf serum (FCS) containing 1–100 mg of an extract of Pelargonium sidoides roots, referred to as EPss 7630 (from Dr. Willmar Schwabe Pharmaceuticals, Karlsruhe, Germany). The extract was dissolved in 12% ethanol and sterilised with a 0.2 mm filter. After 48 h of microaerophilic incubation at 37 1C the turbidity of the medium was monitored at 600 nm. Antibacterial activity of amoxicillin (Sigma, Munich, Germany) was tested by E test. Columbia Agar plates were inoculated with 108 H. pylori by using a sterile swab to cover the plate surface. E test strips were added and the plates were incubated for 48 h under microaerophilic conditions.
During two days incubation in BHI broth the H. pylori count increased from 1 107 to 2.970.04 107 cfu. EPss 7630 (1–50 mg/ml) did not inhibit H. pylori growth. At 100 mg/ml H. pylori growth was reduced to 43710% of control value (po0.05).
Testing for antibacterial activity in coculture with AGS cells
Effect of EPss 7630 and amoxicillin on H. pylori count adherent to AGS cells
The gastric cancer cell line AGS (ATCC CRL 1739) was plated at a density of 104 cells in 96-well-plates in 0.2 ml RPMI 1640 with 10% FBS at 37 1C in a humified atmosphere of 5% CO2 in air and left to adhere for 24 h. Subsequently, cell monolayers were washed with RPMI 1640 and were infected with 105 H. pylori in 0.2 ml medium (70% RPMI 1640, 20% BHI, 10% FCS). After
During the 3 h infection period approximately 30% of the bacteria (3 104 cfu) attached to AGS cells. During the following 21 h incubation period H. pylori density increased to 6–7 105 cfu, i.e. the bacteria replicate. The effects of EPss 7630 and amoxicillin on H. pylori density are shown in Figs. 1 and 2. EPss 7630 (50 and 100 mg/ml) significantly reduced H. pylori density at
Results Inhibition of H. pylori growth by EPss 7630 in BHI medium
ARTICLE IN PRESS W. Beil, P. Kilian / Phytomedicine 14 (2007) SVI 5–8
100
60 *
*
0 0
5
10 50 EPs® 7630 (µg/ml)
100
Fig. 1. Effect of EPss 7630 on H. pylori density attached to AGS cells. AGS cell monolayers were infected with 105 H. pylori. After 3 h cell monolayers were washed to remove nonadherent bacteria and EPss 7630 was added. After 21 h incubation H. pylori density was determined by urease test. Data are expressed as percentage of control (mean7SD of three experiments with Hp 4, 39 and 94). *po0.05 vs. control.
*
*
50
0
0
50 EPs® 7630 (µg/ml)
100
Fig. 3. Effect of EPss 7630 on H. pylori adherence to AGS cells. AGS cell monolayers were infected with 5 106 FITClabelled H. pylori in the presence of EPss 7630. Cell monolayer were washed after 3 h to remove non-adherent bacteria and bacterial adherence was quantified by fluorescence. Data are expressed as percentage of control (mean7SD of three experiments with Hp 4, 39 and 94). *po0.05 vs. control.
EPss 7630 (50 and 100 mg/ml) significantly reduced H. pylori adherence to AGS cells.
100 % H. pylori count
(%) H. pylori count
% H. pylori count
120
7
Discussion
50
0 0
8
16 32 Amoxicillin (µg/ml)
64
Fig. 2. Effect of amoxicillin on H. pylori density attached to AGS cells. AGS cell monolayers were infected with 105 H. pylori. After 3 h cell monolayers were washed to remove nonadherent bacteria and amoxicillin was added. After 21 h incubation H. pylori density was determined by urease test. Data are expressed as percentage of control (mean7SD of three experiments with Hp 4, 39 and 94).
AGS cells to 2375% and 18.775% of control value, respectively. Amoxicillin failed to block H. pylori growth. It should be noted that all H. pylori strains used were susceptible to amoxicillin as determined by E test (data not shown).
EPss 7630 reduces H. pylori adherence to AGS cells To investigate whether EPss 7630 inhibits the adhesion of H. pylori to AGS cells, an in vitro binding assay was used. FITC-labelled H. pylori were overlaid onto AGS cell monolayers in the absence and presence of EPss 7630. After three hours the cell monolayers were washed and the extent of bacteria adhesion was quantified by fluorometric analysis. As shown in Fig. 3
In the present study, we demonstrate that EPss 7630 inhibits (i) H. pylori growth and (ii) reduces bacterial adherence to gastric epithelial AGS cells. A suppressive effect of EPss 7630 against H. pylori growth in BHI broth was observed at a concentration of 100 mg/ml. A significant reduction of H. pylori adherence was observed with EPss 7630 at concentrations X50 mg/ml (Fig. 3). EPss 7630 (50 and 100 mg/ml) reduced density of attached H. pylori during a 21 h incubation period by 77% and 91% (Fig. 2). We, therefore, suggest that the mode of action of EPss 7630 is mainly related to its antiadhesion activity. Bacterial adherence to mucosal surfaces is generally considered to be an important prerequisite for colonisation and infection. H. pylori shows a wide spectrum of different specificities in adhesion to host cells. Several surface carbohydrates mediating cell adhesions have been identified, such as the blood group antigen-binding adhesion (BabA) which recognise the Leb antigen, the sialyl-dimeric-Lewisx glycosphingolid, the sialic acidbinding adhesin (SabA) and sulphated glycosaminoglycans such as heparan sulphate (Lamarque and Peek, 2003; Ruiz-Bustos et al., 2001). It has been reported that sialyllactose, an inhibitor of SabA, significantly reduced the load of the bacteria to gastrointestinal epithelial cells (Simon et al., 1997). Moreover, it was shown that acidic polysaccharides rich of uronic acid from Artemisia capillaris and Panax ginseng inhibited H. pylori adhesion to gastric epithelial cells (Lee et al., 2004).
ARTICLE IN PRESS 8
W. Beil, P. Kilian / Phytomedicine 14 (2007) SVI 5–8
The bioactive ingredients in Pelargonium sidoides are the tri- and tetra-oxygenated coumarins, gallic acid, gallic acid methyl esters and various flavonoids. Pelargonium sidoides contains two distinct coumarins: umckalin and three unique coumarin sulphates (Latte´ et al., 2000). In the present study, we did not test the effect of isolated constituents of the extract on H. pylori adherence. However, it was postulated that negatively charged groups may play a role in host cell–H. pylori adhesion (Lee et al., 2004). Heparan sulphate binding to H. pylori was inhibited by sulphated polysaccharides (heparin and chondroitin sulphates) (Hirmo et al., 1995). Therefore, we suggest that negatively charged components of the extract bind to H. pylori and prevent binding of the bacteria to AGS cells. The antibiotic amoxicillin failed to affect H. pylori growth in co-culture with AGS cells. These results indicate that when H. pylori binds to the AGS cells, the susceptibility to an antibiotic is altered. The concentrations of amoxicillin used were multiples of the MIC values, determined by E test. The mechanism of this resistance is not known. AGS cells are mucous producing cells and form a thin mucous layer which can modify the antimicrobial activity (the biofilm phenomenon). This phenomenon has been shown with H. pylori attached to Hep-2 cells (Me´graud et al., 1991). We conclude that EPss 7630 reduces H. pylori density in vitro by blocking adhesion of H. pylori to gastric host cells.
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