Effect of pomegranate solution alone or combined with chlorhexidine against oral multispecies biofilm

Aim: Natural bioactive products have been tested as alternative antimicrobial agents. This study evaluated the effect of Punica granatum extract (PGE) on oral multispecies biofilms.

Methodology: Lyophilized extracts from pomegranate peel were prepared, and the punicalagin content was assessed by ultra-performance liquid chromatography (UPLC). Oral multispecies biofilms from 2 donors were grown on four collagen-coated hydroxyapatite discs. After incubation for 7 days or 3 weeks, the biofilms were exposed to water (control), 2% CHX, 10% PGE, 20% PGE or 30% PGE for 3 min. The proportions of dead bacteria were assessed by the live/dead staining and confocal microscopy. After the analysis, the best PGE concentration (30%) was combined with CHX. The experimental phases were repeated using water, 2% CHX, 30% PGE and 30% PGE + 2% CHX. Five random areas of the biofilm on each disc were scanned, resulting in 20 scanned areas for each group.

Results: Regarding the biofilm volume, no differences were found amongst solutions (p = .111). The PGE solution killed bacteria effectively in 1-week, 2-week and 3-week-old-plaque biofilms, ranging from 37 to 55.3%, depending on the PGE concentration. The 30% PGE (a) (p = .0009) had greater antibiofilm effectiveness than 2% CHX (b), which killed bacteria in the 25.2 to 48.7% range. The 10% and 20% PGE had intermediate values (ab), without significant differences from 30% PGE (p = 1.002). Water (c) had the lowest proportion of dead bacteria (p < .00001) in a range of 5 to 6.7% and lower effectiveness in killing bacteria (p < .05). The PGE alone or mixed with 2% CHX had greater anti-biofilm effectiveness than CHX (p < .05). The old plaque biofilms were more resistant than the 7-day-old plaque (p < .05).

Conclusions: The 30% PGE (alone or combined with CHX) exhibited a greater antibiofilm effect on oral multispecies biofilms grown on hydroxyapatite discs than 2% CHX.