Mechanism of action of coumarin and silver(I)–coumarin complexes against the pathogenic yeast Candida albicans
Introduction
Coumarin is a a benzopyrone and a naturally occurring constituent of many plants and essential oils, including tonka beans, sweet clover, woodruff, oil of cassia and lavender. Antibiotics containing the coumarin nucleus, such as novobiocin, clorobiocin, and coumermycin A1 produced by a number of the Streptomyces species, were identified over forty years ago. The use of these antibiotics has been limited due to their poor water solubility, low activity against Gram-negative bacteria and the rapid emergence of resistance (Lewis et al., 1996, Laurin et al., 1999). However, renewed interest in these antibiotics has arisen following the discovery that they are potent catalytic inhibitors of DNA gyrase. Additionally, these antibiotics have been shown to be active against Gram-positive bacteria, especially against methicillin-resistant Staphylococcus aureus (MRSA) (Laurin et al., 1999). Further derivatisation of novobiocin, clorobiocin, and coumermycin A1 has allowed for the production of novel coumarin antibiotics displaying excellent inhibition of DNA supercoiling by DNA gyrase B and good antibacterial activity against vancomycin, teicoplanin and novobiocin resistant Enterococci species (Laurin et al., 1999).
Candida albicans is pathogenic yeast which is consistently the most frequently isolated etiological agent of candidosis in humans (Coleman et al., 1998). Candidosis is the commonest invasive fungal infection in patients with malignant haematological disease and in bone marrow transplant recipients (Warnock, 1998). Nosocomial infections due to opportunistic fungal pathogens are a common cause of mortality among hospitalised patients (Micheal, 1995). The development of azole-based anti-fungal drugs has revolutionized the treatment of many fungal infections, but therapy may still necessitate application of the highly toxic drug amphotericin B or a combination of drugs.
Plant extracts containing coumarin derivatives demonstrate anti-fungal activity (Tiew et al., 2003) and some synthetic coumarin derivatives are also active against the yeast C. albicans (Zaha and Hazem, 2002). The presence of phenolic, hydroxy and carboxylic acid groups on the coumarin nucleus has been considered necessary for antimicrobial activity (Kawase et al., 2001). Coumarin derivatives are able to coordinate a transition metal ion via the oxygen of the carbonyl group on the lactone ring (Irena et al., 2001) which raises the possibility that coordinating metals to coumarin may potentiate its anti-microbial toxicity.
The aim of the work presented here was to investigate the anti-fungal activity of some coumarin derivatives and also the silver(I) complexes of these derivatives.
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Culture conditions
C. albicans ATCC 10231 (obtained from the American Type Culture Collection, Maryland, USA) was maintained on YEPD agar [2% (w/v) glucose (Sigma–Aldrich Chemical Co Ltd. Dublin, Ireland), 2% (w/v) bactopeptone (Difco Laboratories, Detroit, USA), 1% (w/v) yeast extract (Oxoid Ltd., Basingstoke, England), 2% (w/v) agar] plates, sub-cultured every 6–8 weeks and stored at 4 °C. Fresh cultures were grown at 30 °C in YEPD broth (as above but without agar). All cultures were grown to the stationary phase
Electron microscopy
Yeast cells were grown to the stationary phase in the presence of half-MIC80 levels of each test agent. Primary fixation of yeast cells was carried out in a 3% (v/v) solution of glutaraldehyde in 0.1 M phosphate buffer for 2 h. Secondary fixation was achieved in 0.2% (w/v) osmium tetroxide in 0.1 M phosphate buffer for 1 h. Samples were dehydrated in graded alcohol solutions of 10, 30, 50, 75, 95, and 100% (v/v) for 15 min. Samples were embedded in Agar 100 resin (Agar Scientific Ltd., UK) and
Extraction of DNA from C. albicans
Yeast cells were grown in the presence of test agent at a concentration equivalent to half MIC80, in Antibiotic Medium 3 at 30 °C and 200 rpm, using an orbital shaker. DNA was extracted from cells (4 × 109) as described earlier (Coyle et al., 2004). The integrity of extracted DNA was determined by agarose gel electrophoresis as described (Coyle et al., 2004). Samples were loaded onto an agarose gel and electrophoresed at 80 V for 1 h. DNA bands were visualised by irradiation at 300 nm and
Statistical analysis
All experiments were performed on three independent occasions and results are the mean ± SEM. Statistical analysis was performed using the non-parametric Mann–Whitney test at a 95% confidence interval.
Effects of coumarin derivatives on fungal cells
A series of novel coumarin ligands and their silver(I) complexes (Table 1) were screened for their anti-fungal activity. The MIC80 of each compounds was determined over a concentration range of 500–0.25 μM. Results indicate that both the number and position of functional groups along with the presence of silver on the coumarin nucleus, greatly affected the fungistatic capacity of the coumarin derivatives. This is evident from the MIC80 values presented in Table 1 and include: [Ag(8-OHCca)] (270
Discussion
The work presented here indicates that while CcaH and [Ag(Cca)] demonstrate anti-fungal activity, derivatising these agents with the inclusion of a hydroxy, nitro or phenanthroline ligand, serves to significantly increase their anti-fungal potency. Most of the silver(I)–coumarin derivatives appear to reduce the respiration rate of C. albicans, possibly by disrupting the synthesis of cytochromes in the mitochondrion. Disruption of the mitochondrial cytochrome content of a cell has the potential
Conflict of interest
The authors have no conflicts of interest to declare.
Acknowledgement
This research was supported by the Technological Sector Research Programme, Strand III (2002–2005), under the European Social Fund Operational Programme for Industrial Development. The research was carried out by the Pharma Research and Development Team jointly located at Institutes of Technology, Tallaght and Dublin, and the National University of Ireland, Maynooth, Co. Kildare, Ireland.
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