Introduction
Dalea (Fabaceae) is an exclusively American genus. D. elegans Gillies ex Hook et Arn is the only species of this genus that grows in the province of Córdoba (Argentina). In previous articles we reported the isolation of the prenylated flavanone 2?, 4?-dihydroxy-5?-(1???, 1???-dimethylallyl)-6-prenylpinocembrin (1). (1) In bioactivity studies, this compound showed antibacterial and antifungal properties, even against multiresistant strains.(2,3) In another biological characterization it demonstrated both antioxidant and antiradical activities; it also inhibited mitochondrial ATP syntase and showed toxic effects against human tumor cells. (4)
Recently, we demonstrated the inhibitory effect of 1 on the efflux of Rhodamine 6G (inhibitory concentration 50, IC50 = 119 µM.) in azole-resistant Candida albicans, which overexpressed Cdr1p, Cdr2p and Mdr1p transporters. In the antifungal activity assay, the value of minimum inhibitory concentration (MIC) for 1 was 150 µM, while that for fluconazole was greater than 400 µM. The MIC for fluconazole decreased 400-fold in the presence of 1 at 100 µM, and thus reversion of azole resistance was achieved. (5)
The aim of this work was to evaluate the interaction of 1 and fluconazole against the resistant C. albicans strain. Rhodamine efflux and cell growth sub-inhibitory concentrations of both compounds were combinated and their activity on the cell growth of the mentioned strain was evaluated.
Materials and methods
Extraction and Isolation
Roots from D. elegans were processed for extraction and isolation of 1 and its structure was established by UV, NMR 1H and 13C and MS, according to Caffaratti et al. (1)
Microorganisms
Two strains of Candida albicans, isolated from the oral cavity, were used. They were a kind gift from Dr. T. White (University of Washington, Seattle, USA). The azole-resistant strain (12-99, hereafter RCa) overexpresses the transporter genes CDR1, CDR2 and MDR1, whereas the sensitive strain (2-76, SCa) lacks these transporter genes. (6) For the experiments, both strains were cultured in yeast peptone dextrose (YPD) broth or Sabouraud agar medium and then resuspended according to the assay performed.
Antifungal activity
To characterize and quantify the antifungal activity, the growth of RCa and SCa were measured in the absence and presence of different concentrations of fluconazole, 1 or their combinations. Absorbance was measured at 540 nm with a MicroQuant microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA). A checkerboard microtiter 96-well plate format was used. Experiments were performed according to the NCCLS-approved standard M27-A (7) as described by Marchetti et al. (8) Minimal inhibitory concentration (MIC) for a compound was defined as the lowest concentration able to produce a growth inhibition higher than 90 % when the viable counts were compared with those of the control conducted in its absence. In a spectrophotometer, it corresponds to the concentration producing an optical density of 50 percent or less with respect to that of the growth control measured at 540 nm in a microplate reader.
Results and discussion
Fluconazole and 1 showed an interaction on the growth of azole-resistant C. albicans at sub-inhibitory concentrations of both. In fact, between 9 and 1 µM, the combination of the two compounds at the same concentration reduced 75-80% growth of yeast as compared to the control measured in absence of the compounds.
The concentration-response curve for fluconazole in the presence of the flavonoid (at a constant concentration of 1 µm) showed inhibitory interaction from 3 µM fluconazole. Similar results were observed in the presence of 3 µM of 1. The presence of the flavonoid reverses the fluconazole resistance of C. albicans; notably, the MIC for the azole is reduced more than 300-fold.
The combination of 9 µM 1 and 700 µM fluconazole is also effective when the concentration of the initial inoculum is 1000-fold higher (10 6 cfu/ml). This effect reduces by 35% cell growth and is observed between 18 and 24 h of incubation of the culture at 37 ºC. The concentration-response curve with this inoculum allows to estimate a 50% growth inhibition for the combination of 1000 µM fluconazole with 9 µM flavonoid while a concentration of 1000 µM of these compounds separately does not affect the growth. The time course of growth inhibition shows that neither 1, fluconazole nor their combination has any effect during the first hour of incubation and the flavonoid presents its maximum inhibitory effect between 18 and 24 h.
Conclusion
In a previous work we demonstrated that1 inhibits the Cdr1p azole transporters present in fluconazole-resistant C. albicans; it acts in a concentration-dependent manner with an IC50 = 119 µM. (5) This compound also inhibits the growth of this strain with a MIC of 150 µM. Besides, we showed that MIC for fluconazole decreased by more than 400-fold in the presence of 1 at 100 µM. (5)
In order to avoid the per se inhibitory effect of these compounds, in this opportunity we evaluated the interaction between sub-inhibitory concentrations of the azole and the flavonoid. Growth inhibition with concentrations 100-fold smaller than the MIC for the flavonoid and more than 400-fold smaller than the MIC for fluconazole was observed.
Since the concentrations of 1 tested in this work are much lower than the IC50 of the Cdr1p transporters, and practically inactive on them, the results suggest that other mechanisms could be implicated in the inhibitory interaction of 1 with fluconazole. This effect would also occur with yeast inoculum between 103 and 106 cfu/ml and has the maximum values at 24 h of incubation.
The facts that ATP provides the energy for the transport, and the mitochondrial ATPsintase of other cells was inhibited by 1, (4) suggest the hypothesis that a similar effect may be involved in resistant C. albicans. We are currently carrying out studies in order to check it.
References
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2) Ortega M G, Scarafia ME, Juliani HR. Antimicrobial Agents in Dalea elegans. Fitoterapia 1996; 67: 81.
3) Pérez C, Tiraboschi IN, Ortega MG, Agnese AM, Cabrera JL. Further antimicrobial studies of 2?, 4?-dihidroxy-5?-(1???,1???-dimethylallyl)-6-prenylpinocembrin from Dalea elegans. Pharm. Biol. 2003, 41:171.
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7) National Committee for Clinical and Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of yeasts. In: Approved standard M27-A. Villanova, Pa; 1997. 17.
8) Marchetti O, Moreillon P, Glauser M, Bille J, Sanglard D. Potent synergism of the combination of fluconazole and cyclosporine in Candida albicans. Antimicrob Agents Chemother. 2000; 44: 2373-2381.
Acknowledgements
This work was supported by FONCyT: BID PICT No.1576, CONICET and CONAMED Foundation