Synergistic activity of pomegranate rind extract and Zn (II) against Candida albicans under planktonic and biofilm conditions, and a mechanistic insight based upon intracellular ROS induction

Abstract

Candida albicans (C. albicans) is an opportunistic pathogen, which causes superficial infection and can lead to mortal systemic infections, especially in immunocompromised patients. The incidence of C. albicans infections is increasing and there are a limited number of antifungal drugs used in treatment. Therefore, there is an urgent need for new and alternative antifungal drugs. Pomegranate rind extract (PRE) is known for its broad-spectrum antimicrobial activities, including against C. albicans and recently, PRE and Zn (II) have been shown to induce synergistic antimicrobial activity against various microbes. In this study, the inhibitory activities of PRE, Zn (II) and PRE in combination with Zn (II) were evaluated against C. albicans. Antifungal activities of PRE and Zn (II) were evaluated using conventional microdilution methods and the interaction between these compounds was assessed by in vitro checkerboard and time kill assays in planktonic cultures. The anti-biofilm activities of PRE, Zn (II) and PRE in combination with Zn (II) were assessed using confocal laser scanning microscopy, with quantitative analysis of biofilm biomass and mean thickness analysed using COMSTAT2 analysis. In addition, antimicrobial interactions between PRE and Zn (II) were assayed in terms reactive oxygen species (ROS) production by C. albicans. PRE and Zn (II) showed a potent antifungal activity against C. albicans, with MIC values of 4 mg/mL and 1.8 mg/mL, respectively. PRE and Zn (II) in combination exerted a synergistic antifungal effect, as confirmed by the checkerboard and time kill assays. PRE, Zn (II) and PRE and Zn (II) in combination gave rise to significant reductions in biofilm biomass, although only PRE caused a significant reduction in mean biofilm thickness. The PRE and Zn (II) in combination caused the highest levels of ROS production by C. albicans, in both planktonic and biofilm forms. The induction of excess ROS accumulation in C. albicans may help explain the synergistic activity of PRE and Zn (II) in combination against C. albicans in both planktonic and biofilm forms. Moreover, the data support the potential of the PRE and Zn (II) combination as a novel potential anti-Candida therapeutic system.

Characterization, Antimicrobial Activity, and Antioxidant Efficacy of a Pomegranate Peel Solution Against Persistent Root Canal Pathogens

“…The UPLC test showed the presence of α-punicalagin and β-punicalagin with two almost similar peaks in the UV spectrum compared to the positive control. This result corroborates with the findings by Celiksoy et al, who found two similar punicalagin peaks in the UPLC chromatogram [ 16 ]. However, the total amount of phenolic content was different between researchers.…”

“…The polyphenolic and tannin compounds (as the punicalagin) promote the antimicrobial activity of the PGE through the precipitation of proteins on the cell surface of the microorganism and inhibiting metabolic enzymes that cause cell lysis [ 16 , 28 ]. The preparation method and chemical characterization of tested extracts are essential because their activity is directly affected by the plant part, plant age, season of collection, drying strategy, and extraction procedure [ 3 ].…”

“…Determination of the punicalagin content in the PGE was carried out by UPLC using pure punicalagin as the standard (Sigma-Aldrich, St Louis, USA) [ 16 ].…”

“…Previous studies have evaluated the antimicrobial and antioxidant activities of pomegranate peel extracts [ 4 , 8 , 13 , 16 , 17 , 25 ], including two studies that tested it inside the root canal system as an intracanal medicament [ 4 , 8 ]. Most studies have used an aqueous extract instead of hydroalcoholic [ 4 , 8 , 17 ], which can compromise the amount of phytochemicals in the solution.…”

“…Some still need to perform the chemical characterization of the extract [ 4 , 8 , 25 ], which can directly influence their pharmacological activities [ 26 ]. Others did not evaluate the pomegranate solution against persistent root canal pathogens [ 16 , 17 , 25 ].…”