Candida species are now the fourth most common organism recovered from the blood of hospitalized patient (Gudlaugsson et al. 2003). Notwithstanding the increasing need for effective therapy, the range of antifungal agents available is limited, and some of the most effective agents are also toxic. In addition, while the azoles have been used successfully for the treatment of Candida infections, numerous reports of treatment failures are now appearing in the literature (Lipsitch et al. 2000; Perea and Patterson 2002).

Previous reports of synergistic combinations of antifungal compounds have not been extensive or encouraging. In a review of combination therapy in systemic mycosis, Polak (1990) discussed the combination of amphotericin B with flucytosine as one of the better-established synergistic combinations of antifungal agents that has been used clinically to treat candidiasis. The combination of an azole with a polyene resulted in conflicting outcomes, depending on the species and the strain tested, and specific antagonism was observed with Candida albicans (Polak 1990).

Berberine, a well-known alkaloid, was found in medicinal herbs such as Coptis chinensis Franch (Ranunculaceae) and Hydrastis canadensis L. (Ranunculaceae). Currently, the predominant clinical uses of berberine preparations include the treatment of bacterial diarrhea, intestinal parasite infections and ocular trachoma infections (Birdsall 1997). Berberine was demonstrated to have weak activity against C. albicans and C. glabrata (Polak 1990; Park et al. 1999; Vollekova et al. 2003). Other pharmacological effects of berberine, such as antiarrhythmic (Zhao and Guo 1989), anti-inflammatory (Kuo et al. 2004), anticancer (Mitani et al. 2001) immunosuppressive (Marinova et al. 2000), vasorelaxant, and antiproliferative (Ko et al. 2000) have also been reported.

The main aim of the study described here was to investigate ways of enhancing the antifungal activities of the azoles. We have focused on combination of berberine with fluconazole, because fluconazole is one of the most popular drugs for treating candidiasis, especially in immunocompromised patients.

C. albicans ATCC 10231 and clinical isolates from vaginal fluid were tested by disk diffusion test (Marchetti et al. 2000). A 106 CFU/ml yeast suspension was applied onto the yeast extract-peptone-dextrose (YEPD) agar plate with and without 6 μg/ml of fluconazole using cotton swabs. Then, for C. albicans ATCC 10231, paper disks impregnated with 0, 1, 10, 100, and 500 μg of berberine were placed onto agar plates. For isolates, berberine was tested at 0, 10 and 100 μg/disk. Diameters of growth inhibition zones were measured after incubation at 37°C for 24 h.

Checkerboard tests were performed by broth microdilution reference procedure at a final inoculum of 0.5–2.5 × 103 CFU/ml, using RPMI 1640 medium buffered with 0.165 M MOPS (CLSI 2004). The final concentrations ranged from 0.06 to 62.5 μg/ml for fluconazole and 1.9 to 125 μg/ml for berberine. Plates were incubated at 37°C for 48 h and absorbance was read in a ELISA plate reader at 492 nm. The fractional inhibitory concentration (FIC) index is the sum of the MIC of each drug in combination divided by the MIC of the drug used alone. An FIC index ≤0.5 is considered synergism; FIC index >4 is antagonism and a result >0.5 but ≤4 is indifferent (Odds 2003).

In order to investigate the effect of the berberine and fluconazole alone and in combination on morphology of C. albicans, treated and control cells were examined by scanning electron microscopy. Treated and untreated cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2 and small drops of the fixed cells were placed on specimen support with poly-l-lysine for 1 h at room temperature. Subsequently, the samples were dehydrated in graded ethanol, critical-point dried in CO2, coated with gold and examined in a Shimadzu SS-550 scanning electron microscope.

The alkaloid berberine is a common component of a variety of plant species, particularly in the family Berberidaceae. Berberine exhibits relatively weak antibiotic properties, apparently because of its efflux by multidrug resistance pumps (Stermitz et al. 2001).

In the present study, combinations of berberine and fluconazole were tested by an agar disk diffusion assay in which clinical isolates of C. albicans were applied onto YEPD agar plate. Agar diffusion tests visualized a synergistic interaction. Berberine had no antifungal activity in smaller amounts and showed a weak antifungal activity at 500 μg (Fig 1a). Fluconazole at 6 μg/ml had no antifungal activity. In contrast, berberine showed a powerful fungistatic effect on the agar plate containing 6 μg/ml fluconazole (Fig. 1b).

Fig. 1
figure 1

Disk diffusion halo assays demonstrate enhanced inhibition of C. albicans ATCC 10231 when berberine is combined with fluconazole. A 106 CFU/ml yeast suspension was applied onto the yeast extract-peptone-dextrose (YEPD) agar plate without (a) and with (b) 6 μg/ml of fluconazole using cotton swabs. Paper disks impregnated with 0, 1, 10, 100 and 500 μg of berberine were placed onto agar plates. Diameters of growth inhibition zones were measured after incubation at 37°C for 24 h

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To quantify the antifungal activities obtained with berberine alone and in combination with fluconazole, antifungal indices were calculated. An increase of the antifungal index reflected a synergistic effect. Table 1 summarizes the in vitro susceptibilities of 25 isolates of C. albicans as determined by disk diffusion testing. Berberine alone had in this experimental setting a low intrinsic antifungal activity (median 0.04; range 0–0.2), and its combination with fluconazole had a powerful antifungal activity with a median index of 1.26 (range 0–2.1).

Table 1 Agar disk testing: antifungal indices of Berberine tested alone and in combination with fluconazole (FLC) against clinical isolates of Candida albicans
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Berberine and fluconazole were also tested by the checkerboard assay to determine whether they had favorable or unfavorable antifungal interactions. The MIC of fluconazole was 1.9 μg/ml when the drug was tested alone and decreased to 0.48 μg/ml in the presence of berberine concentrations of 1.9 μg/ml. However, berberine at concentrations of >1.9 μg/ml combined with a fluconazole supra-MIC (i.e., >1.9 μg/ml) eliminated the residual turbidity (trailing) in the incubation wells. This observation made it possible to use a different endpoint, the MIC-0. The MICs-0 for yeast were graphed in isobolograms and yielded highly concave curve, characteristic of strong drug synergism (data not shown).

Electron micrographs were taken to determine if any structural changes took place in C. albicans after treatment with berberine alone and in combination with fluconazole (Fig. 2). The control cells of C. albicans presented a smooth surface and sub-spherical or oval shape, with a great capacity for producing blastospores (Fig. 2a). After exposure to berberine alone, the general aspect of the cells was not modified; although, some of the yeast cells began to cluster (Fig 2b). Cells exposed to fluconazole alone were rounder and showed a tendency to agglutinate; lemon-shaped yeasts with small buds were still present, but we also observed a great capacity for producing pseudohyphae (Fig. 2c). Cells exposed to berberine in combination with fluconazole were collapsed and showed deep surface folds. Some of them also showed clear degradations and appeared surrounded by cellular debris (Fig. 2d).

Fig. 2
figure 2

Scanning electron microscopy photographs of C. albicans ATCC 10231 treated with berberine alone and in combination with fluconazole. a Control; b berberine 10 μg/ml; c fluconazole 6 μg/ml; and d berberine plus fluconazole. Bar = 10 μm

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The results presented here indicate a strong synergistic interaction between berberine and fluconazole in inhibiting the growth of C. albicans. These drugs had no intrinsic antifungal activity at the concentration tested. By checkerboard microtiter testing it was observed that supra-MIC concentrations of fluconazole had only a weak fungistatic effect, and this finding correlated well with the measured residual turbidity, the so-called trailing. However, above a given minimal concentration, berberine eliminated completely the trailing effect when combined with supra-MIC of fluconazole. The term trailing has been used to describe the reduced but persistent growth that some isolates of Candida sp. exhibit over an extended range of drug concentrations. For some isolates, however, trailing growth is so significant that the MICs for these isolates will appear to be low after 24 h but much higher after 48 h. These MICs are so discordant that they place the isolate into different MIC interpretive categories at the two time points. The reduction of trailing observed in the present study was explained by a strain-dependent potent fungistatic or fungicidal effect resulting from the combination of berberine with fluconazole. Since this effect decreased the viable counts by >99.9% compared to those of the growth control, a different endpoint, the MIC-0, was chosen.

Prolonged administration of fluconazole in immunocompromised patients can result in the appearance of azole-resistant clinical isolates over expressing multi-drug efflux transporters including ABC transporters or major facilitator super family (MFS) transporters (White et al. 1998). Suppressing the activity of these fungal ABC transporters with small molecule multi-drug efflux pump inhibitors could reduce the drug resistance of these pathogenic fungi and therefore help to increase the efficacy of antifungal chemotherapy with triazoles. Berberine is a weak antimicrobial produced by a wide variety of plant species. It is an amphipathic cation that resembles quaternary ammonium antiseptics in its chemical properties and possibly in its mechanism of action as well. The likely targets of berberine are the cytoplasmic membrane and DNA, into which it intercalates (Jennings and Ridler 1983). Amphipathic cations are the preferred substrates of most multidrug resistance pumps (Lewis 2001) Several studies have shown that plants of the genus Berberis (Berberis repens, B. aquifolium and B. fremontii) producing berberine synthesise two substances, the flavonolignan 5′-MHC-D and the porphyrin pheophorbide a, which have no antibacterial activity but have an inhibiting property against MDR efflux pumps found so far in Staphylococcus aureus (allowing berberine to carry out its activity) (Stermitz et al. 2000a, b, 2001). In the absence of efflux, berberine, a hydrophobic cation, accumulates in the cells of microbial pathogens, and the accumulation is driven by the membrane potential (Severina et al. 2001).

Since the berberine concentration effective in vitro is achievable in vivo, the combination of this agent with fluconazole represents an attractive perspective for the development of new management strategies for candidiasis.