A Prenylated Flavanone from Dalea elegans Inhibits Rhodamine 6 G Efflux and Reverses Fluconazole-Resistance in Candida albicans

Abstract

In previous studies, 2',4'-dihydroxy-5'-(1''',1'''-dimethylallyl)-6-prenylpinocembrin, a prenylated flavonoid isolated from Dalea elegans roots, showed activity against multiresistant Staphylococcus aureus and Candida albicans, as well as an uncoupling effect on mitochondria and antioxidant activity. The aim of this study was to evaluate the inhibitory effects of 2',4'-dihydroxy-5'-(1''',1'''-dimethylallyl)-6-prenylpinocembrin and fluconazole on the efflux of rhodamine 6 G in azole-resistant C. albicans 12-99 that expresses multidrug transporters Cdr1p, Cdr2p, and Mdr1p. The effect of fluconazole and 2',4'-dihydroxy-5'-(1''',1'''-dimethylallyl)-6-prenylpinocembrin on rhodamine 6 G efflux was assessed in both azole-sensitive and azole-resistant C. albicans. Between 1 and 1000 µM, 2',4'-dihydroxy-5'-(1''',1'''-dimethylallyl)-6-prenylpinocembrin inhibited rhodamine 6 G efflux only in azole-resistant C. albicans 12-99 in a concentration-dependent manner (IC50 = 119 µM); a competitive effect was observed. It also showed selectivity of action in comparison with other flavanones (6-prenylpinocembrin, isolated from aerial parts of D. elegans, pinocembrin, naringenin, and hesperetin, all at 250 µM). To check the possible implications of the inhibition of azole efflux on cell growth, antifungal assays were conducted. Minimal inhibitory concentration values were 150 µM for 2',4'-dihydroxy-5'-(1''',1'''-dimethylallyl)-6-prenylpinocembrin and higher than 400 µM for fluconazole. The combination of both compounds at either inhibitory or subinhibitory concentrations was significantly more effective than each compound separately. Minimal inhibitory concentration for fluconazole decreased by more than 400 times in the presence of 100 µM 2',4'-dihydroxy-5'-(1''',1'''-dimethylallyl)-6-prenylpinocembrin, reversing azole resistance and giving values similar to those of azole-sensitive C. albicans. These data are consistent with a dual action of 2',4'-dihydroxy-5'-(1''',1'''-dimethylallyl)-6-prenylpinocembrin: direct antifungal effect on azole-resistant C. albicans 12-99 and inhibition of azole transporters, which results in reversion of fluconazole resistance.

The roles of CDR1, CDR2, and MDR1 in kaempferol-induced suppression with fluconazole-resistant Candida albicans

“…The upregulations of multidrug efflux pump controlled by Cdr1p, Cdr2p belonging to ATP-binding cassette superfamily (APC transporter) and Mdr1p, a member of major facilitator superfamily (MFS) were implicated in most fluconazole-resistant C. albicans strains (Niimi et al 2004;Holmes et al 2012;Prasad and Rawal 2014) as FLC was a substrate for CDR1, CDR2, and MDR1 (Kohli et al 2001;Nakamura et al 2001). Rhodamine 6G was the most common used fluorescent dye of ABC pump substrate requiring ATP as energy to observe transporter-mediated FLC resistance (Lamping et al 2007;Peralta et al 2012). Our experiments showed that FLC alone failed to lead to rhodamine 6G accumulation in the intracellular space in C. albicans z2003 due to active efflux pump (Figure 3).…”

“…The pellets were washed three times by sterile PBS. As efflux of rhodamine-6G from Candida cells required the presence of glucose, glucose dissolved in PBS was coincubated with the pellets to the final concentration of 2 mmol/L at 37 C 200 rpm for 1 h. The suspension was centrifuged at 3000 g. An aliquot of supernatant (100 mL) was removed and detected at an excitation wavelength of 525 nm and an emission wavelength of 550 nm with an inverted fluorescence microscope IX71 (Olympus, Tokyo, Japan) (Peralta et al 2012). …”

“…Flavonoids are a group of chemicals, and have also been reported to have broad-spectrum antibacterial and antifungal activities (Friedman 2007;Orhan et al 2010;Peralta et al 2012). Kaempferol (KAE), one of the active flavonoids, widely exists in many fruits and plants with broad-spectrum antioxidant, anti-inflammatory and anticancer properties (Rajendran et al 2014).…”

Flavanoids from Dalea elegans: Chemical reassignment and determination of kinetics parameters related to their anti-tyrosinase activity

“…1). These prenylated derivatives of pinocembrin have proven to be highly active in different biological activity assays (Ortega et al, 1996;Pé rez et al, 2003;Elingold et al, 2008;Peralta et al, 2012). Another species of Dalea was studied in our group, D. boliviana, and showed the presence of new prenylated flavanones active as tyrosinase inhibitors (Peralta et al, 2011).…”

The Natural Flavonoid Pinocembrin: Molecular Targets and Potential Therapeutic Applications

“…In the decades since, studies have shown that pinocembrin can significantly inhibit the activity of Penicillium italicum [60], Candida albicans [61–65], Staphylococcus aureus [66,67,61,68,69,65,11], E. coli [70,61], Bacillus subtilis, Trichophyton mentagrophytes, Streptococcus mutans [71,65,19], and Neisseria gonorrhoeae [72]. Recently, Soromou et al [66] reported that pinocembrin reduced α-haemolysin production and attenuated α-haemolysin-mediated cell injury at low concentrations and protected mice from S. aureus -induced pneumonia.…”