Antibacterial and Time-Kill Evaluation of Punica Granatum Against Cariogenic Bacteria

Objectives: Punica granatum (PG), commonly known as pomegranate, is highly accessible and widely renowned for its variety of therapeutic effects. However, not enough information is available concerning its antimicrobial effect against cariogenic microorganisms. The aims of this study are to investigate the antimicrobial activity of commercially available PG extract and the time for total inhibition of caries-associated bacteria; as well as to identify the active ingredient responsible for this antibacterial action.
Methods: Standard strains of Streptococcus mutans (DMST18777), Streptococcus sanguis (DMST18782) and Lactobacillus casei (BCC36987) were submitted to time-kill assay. PG extract dissolved in water at 500mg/mL concentration was inoculated with a suspension of each bacterial strain separately. To determine the time-kill, 100uL of mixture from each exposure time (0hr, 1hr, 6hr, 8hr, 12hr and 24hr) were taken, neutralized and diluted 10-fold with PBS. In triplicate, 10uL of the diluted samples were dispensed in selective culture agar and incubated at 37^oC for 24hrs. The total viable colonies (CFU/mL) were counted and log₁₀ reductions were determined for each time point as compared to negative control. In addition, phytochemical analyses for identification of active ingredient were carried out by High Performance Liquid Chromatography (HPLC).
Results: For time-kill assay of all strains, there is an increase in reduction of total viable colonies with time as compared to population control. There was total inhibition of S. mutans at 6hrs; S. sanguis at 1hr. and L. casei at 24hrs. HPLC results showed presence of two punicalagin isomers at average retention time of 16.8min and 21.3min.
Conclusions: Aqueous PG extract at 500mg/mL concentration demonstrate antibacterial effects against cariogenic bacteria possibly due to the activity of its biomarker, punicalagin; Thus, it has the potential to be a new anti-caries agent.