# Antibacterial Effects of _Paeonia lactiflora_ Extract on Oral Microcosm Biofilms
## 4 Discussion  
In this study, PL extract exhibited antibacterial effects on oral microcosm biofilms that were comparable to those of CHX, as evaluated by measurements of red fluorescence intensity, bacterial viability, and aciduric bacterial counts. Before assessing the antibacterial effects of PL extract, we evaluated its cytotoxicity to determine the optimal treatment concentration. The results showed that the cell viability at a concentration of 30 \(\upmu\)g/mL was comparable with that at 50, 100, and 150 \(\upmu\)g/mL, with all concentrations exhibiting over 70% cell viability; this confirmed the absence of cytotoxicity (Table 1). Therefore, despite the absence of cytotoxicity at all tested concentrations, we set the PL extract concentration at 30 \(\upmu\)g/mL to minimize potential cytotoxicity and enhance cost-effectiveness. The red fluorescence intensity observed in this study primarily reflects the accumulation of endogenous porphyrins and EPSs [38; 39], which are integral components of mature oral biofilms. In addition, this indicator may also be influenced by the dynamic interactions within the oral biofilm community, including bacterial communication and metabolic activity. The analysis of RatioR/G in the oral microcosm biofilms revealed similar effects of PL extract and CHX in terms of inhibiting the maturation of the oral microcosm biofilms (Figure 2). This finding was in contrast to that in a study reporting that PL extract, when compared with CHX, exhibited superior antimicrobial effects against _C. albicans_ biofilms [40]. This discrepancy may be attributed to the significantly lower concentration used in the present study (30 \(\upmu\)g/mL) than in the previous study (250-4000 \(\upmu\)g/mL), and to differences in the target microorganisms (_C. albicans_ vs. oral microbiota). Because this study evaluated a low-concentration PL extract using an oral microcosm biofilm model that closely mimics the actual oral environment, it can be considered a study design that is more reflective of clinical settings than are other study designs. Another possible reason is that the oral microcosm biofilm may have hindered the penetration of CHX more effectively than the single-species biofilm; this is because multispecies biofilms have more \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Treatment** & **N** & **Live Bacteria** & **Dead Bacteria** &
\begin{tabular}{c} **Bacterial Viability** \\ **(RatioG/G+R)** \\ \end{tabular} \\ \hline Distilled water & 8 & 12.01 (3.70) 1  & 9.36 (2.60) 1  & 0.56 (0.09) 1  \\ _Paemia lactisiflora_ & 8 & 6.33 (0.67) 1  & 10.64 (5.06) 1  & 0.41 (0.11) 2  \\ Chlorexidine & 8 & 6.03 (1.09) 1  & 9.71 (2.47) 1  & 0.39 (0.06) 1  \\ _p_-values 2  & 0.006 & 0.845 & 0.003 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Bacterial viability in oral microcosm biofilms after treatment. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Treatment** & **N** & **logCFU/mL** \\ \hline Distilled water & 14 & 6.38 (0.12) 1  \\ _Paemia lactisiflora_ & 14 & 6.18 (0.13) 2  \\ Chlorexidine & 14 & 6.16 (0.12) 3  \\ _p_-values 4  \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Aciduric bacterial counts after treatment. complex structures and bacterial interactions, which increase their resistance to antimicrobial agents [41]. In this study, both PL extract and CHX similarly inhibited the maturation of oral microcosm biofilms, as evidenced by the reduced red fluorescence intensity, which reflects decreased biofilm maturity and activity [42]. This effect may be attributed to the down-regulation of the expression of glucosyltransferase, which promotes the accumulation of EPSs, by the polyphenols and flavonoids in PL extract [43]. This downregulation was also reported to decrease the synthesis of polysaccharides secreted by _S. mutans_, consequently reducing the pathogenicity of the oral biofilm [43]. Our previous studies have comprehensively analyzed the chemical composition of PL extract, identifying polyphenols and flavonoids as its key active components [29,44].