Hinokitiol Preventing Porphyromonas gingivalis from Forming Biofilm and Producing Gingipains via Quorum Sensing Inhibition

The aim of this study is to verify that hinokitiol suppresses biofilm formation and gingipain production by inhibiting quorum sensing in Porphyromonas gingivalis. Periodontal disease is a chronic inflammatory condition caused by the colonization of P. gingivalis, which form biofilms and produce virulence factors such as gingipains. It is known that the formation of biofilms and the production of gingipains in P. gingivalis are regulated by quorum sensing via autoinducer-2 (AI-2). In this study, we hypothesized that hinokitiol, a natural disinfectant, can inhibit quorum sensing in P. gingivalis at sublethal concentration, thereby suppressing biofilm formation and gingipain production, leading to therapeutic effects against periodontal disease. AI-2 bioassay with Vibrio harveyi revealed that 20–300 µM hinokitiol inhibited V. harveyi luminescence caused by AI-2. Furthermore, 100 µM hinokitiol inhibited the AI-2 activity in a culture supernatant of P. gingivalis. Therefore, we evaluated whether hinokitiol at sublethal concentrations suppressed biofilm formation and gingipain production. A 96-well plate biofilm assay showed that 70–100 µM hinokitiol suppressed biofilm biomass. Moreover, the suppression of biofilm formation reduced resistance of P. gingivalis to hinokitiol. Western blotting analysis revealed that the expressions of membrane-bounded Arg-gingipain and secreted Arg-gingipain and Lys-gingipain were suppressed by 30 µM of hinokitiol. These findings suggested that hinokitiol suppress biofilm formation and gingipain production by inhibiting P. gingivalis quorum sensing via AI-2.