Elsevier

Toxicology in Vitro

Volume 21, Issue 5, August 2007, Pages 801-808
Toxicology in Vitro

Mechanism of action of coumarin and silver(I)–coumarin complexes against the pathogenic yeast Candida albicans

https://doi.org/10.1016/j.tiv.2007.01.022Get rights and content

Abstract

The anti-fungal activity and mode of action of a range of silver(I)–coumarin complexes was examined. The most potent silver(I)–coumarin complexes, namely 7-hydroxycoumarin-3-carboxylatosilver(I), 6-hydroxycoumarin-3-carboxylatosilver(I) and 4-oxy-3-nitrocoumarinbis(1,10-phenanthroline)silver(I), had MIC80 values of between 69.1 and 4.6 μM against the pathogenic yeast Candida albicans. These compounds also reduced respiration, lowered the ergosterol content of cells and increased the trans-membrane leakage of amino acids. A number of the complexes disrupted cytochrome synthesis in the cell and induced the appearance of morphological features consistent with cell death by apoptosis. These compounds appear to act by disrupting the synthesis of cytochromes which directly affects the cell’s ability to respire. A reduction in respiration leads to a depletion in ergosterol biosynthesis and a consequent disruption of the integrity of the cell membrane. Disruption of cytochrome biosynthesis may induce the onset of apoptosis which has been shown previously to be triggered by alteration in the location of cytochrome c. Silver(I)–coumarin complexes demonstrate good anti-fungal activity and manifest a mode of action distinct to that of the conventional azole and polyene drugs thus raising the possibility of their use when resistance to conventional drug has emerged or in combination with such drugs.

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.

Section snippets

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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