Novel Bi-Factorial Strategy against Candida albicans Viability Using Carnosic Acid and Propolis: Synergistic Antifungal Action

Abstract

The potential fungicidal action of the natural extracts, carnosic acid (obtained from rosemary) and propolis (from honeybees’ panels) against the highly prevalent yeast Candida albicans, used herein as an archetype of pathogenic fungi, was tested. The separate addition of carnosic acid and propolis on exponential cultures of the standard SC5314 C. albicans strain caused a moderate degree of cell death at relatively high concentrations. However, the combination of both extracts, especially in a 1:4 ratio, induced a potent synergistic pattern, leading to a drastic reduction in cell survival even at much lower concentrations. The result of a mathematical analysis by isobologram was consistent with synergistic action of the combined extracts rather than a merely additive effect. In turn, the capacity of SC5314 cells to form in vitro biofilms was also impaired by the simultaneous presence of both agents, supporting the potential application of carnosic acid and propolis mixtures in the prevention and treatment of clinical infections as an alternative to antibiotics and other antifungal agents endowed with reduced toxic side effects.

A Specific Mixture of Propolis and Carnosic Acid Triggers a Strong Fungicidal Action against Cryptococcus neoformans

“…The fractional inhibitory concentrations index (FICI) calculated from the corresponding MIC 50 was 0.49. This result, together with the isobolograms also performed ( Figure S1 ), provided consistent evidence that the combined mixture of CA and PP3 shows strong synergy [ 10 , 20 ].…”

“…Whereas PP1 was virtually inactive at the concentrations tested, PP2 only displayed conspicuous antifungal action at 1000 µg/mL, and PP3 showed the strongest fungicidal outcome since an intense antifungal action was observed at 500 µg/mL. In turn, CA, even at lower concentrations (50 μg/mL), also displayed a noticeable fungicidal effect against C. neoformans, similar to that previously recorded in C. albicans [ 20 ].…”

“…For instance, mixtures containing propolis plus Amphotericin B or azoles caused a drastic cell killing in C. albicans [ 21 ]. We recently demonstrated that a specific combination of PP3 and CA possesses a strong fungicidal effect against C. albicans , whether in laboratory or in clinical strains [ 20 ]. However, limited information is available on equivalent formulations combining propolis together with other natural compounds on different fungal species.…”

“…The time-course kinetics of cell survival recorded in growing H99 cells revealed that the combinatorial addition of CA and PP3 induced a stronger fungicidal action in comparison with the action exerted by the isolated components at the tested doses. The combination ratio was 1:4 (50 μg/mL CA and 200 μg/mL PP3), which was the optimal ratio already established for C. albicans [ 20 ]. Consistent results were obtained by either direct counting of viable cells in liquid media (CFUs/mL) ( Figure 3 A) or by macroscopic colonial formation on solid plates ( Figure 3 B).…”

“…In the search for new, potentially interesting, antimicrobial designs based on propolis, we assayed the combination of PP and carnosic acid (CA), a bioactive terpenoid extracted from rosemary ( Rosmarinus officinalis ) and endowed with both antioxidant and antimicrobial activities [ 17 , 18 , 19 ]. A new, potent antifungal effect triggered by combinatorial mixtures of CA and PP has recently been demonstrated against clinical isolates of the highly prevalent yeast Candida albicans [ 20 ]. Here, we studied the possible fungicidal action of these formulations against the dangerous and opportunistic basidiomycete C. neoformans , whose capacity to develop in vitro biofilms was also analyzed.…”