Conditions for Preparing Glycyrrhiza uralensis Extract for Inhibiting Biofilm Formation of Streptococcus mutans

Abstract

Licorice, which has an extensive history of use as an herbal medicine, has been suggested to have oral health benefits. However, to date, no systematic study has been conducted on the preparation method of licorice extracts for oral health. In this study, licorice extracts prepared using water and ethanol were investigated for its ability to inhibit the biofilm formation of Streptococcus mutans. The licorice extract prepared with around 60% ethanol effectively inhibited the biofilm formation of S. mutans. Licorice extracted with 50% ethanol almost completely inhibited the biofilm formation at 1.5 g/L of licorice extract. This inhibitory activity was confirmed in a microplate assay and a flow cell system. Glycyrrhetic acid was extracted from licorice effectively with 60% ethanol concentration. The strong inhibitory activity of glycyrrhetic acid and the synergistic inhibition with glycyrrhizin on biofilm formation were suggested as major reasons for a concentration-specific extraction. These results suggest that licorice extract prepared using around 60% ethanol effectively inhibits the biofilm formation of S. mutans.

Fig. 1. Effect of ethanol content of extract solvent on the biofilm formation (A and C) and the cell growth (B and D) of S. mutans. Extracts of 5% (A and B) and 1.25% (B and C) of the tested culture volume were treated. The control value is the result of the untreated extract, which is indicated on the right side of the figure by a black arrow. The standard deviation was calculated from five independent experimental results. Statistical analysis was performed using a tukey test. Values that differ from the control with the 95% confidence level are marked with a star on the top of symbols.

Fig. 2. Effect of the licorice-to-solvent volume ratio on the biofilm formation (A, C, and E) and the cell growth (B, D, and F) of S. mutans. The inhibitory activity of extracts with 5% of tested cell culture volume was evaluated according to the volume of extract solvent: water (A and B), 50% ethanol (C and D), and 95% ethanol (E and F). The control value is the result of the untreated extract, which is indicated on the right side of the figure by a black arrow. The standard deviation was calculated from five independent experimental results. Statistical analysis was performed using a tukey test. Values that differ from the control with the 95% confidence level are marked with a star on the top of symbols.

Fig. 3. Effect of extract amount on the biofilm formation and the cell growth of S. mutans. The biofilm formation (A) and cell growth (B) were evaluated using a microplate assay. The control value is the result at 0 g/L in each figure. The standard deviation was calculated from five independent experimental results. Statistical analysis was performed using a tukey test. Values that differ from the control with the 95% confidence level are marked with a star on the top of symbols.

Fig. 4. Effect of licorice extract on the biofilm formation of S. mutans in a flow cell chamber. The microscope magnification was ×400. Scale bar on the bottom right of figure indicates 50 μm.

Fig. 5. Effect of glycyrrhizin, glycyrrhetic acid, liquiritin, and glucuronic acid on the biofilm formation (A, C, E, and G) and the growth inhibition (B, D, F, and H) of S. mutans. The control value is the result at 0 g/L in each figure. The standard deviation was calculated from five independent experimental results. Statistical analysis was performed using a tukey test. Values that differ from the control with the 95% confidence level are marked with a star on the top of symbols.

Fig. 6. Synergistic inhibition of a mixture of glycyrrhizin and glycyrrhetic acid on the biofilm formation (A) and the cell growth (B) of S. mutans. The concentrations were 1.25 mM for glycyrrhizin (GLZ) and 0.075 mM for glycyrrhetic acid (GA). The control value is the result of 5% treatment of DMSO, a solvent in which the compound is dissolved, without any compound. The standard deviation was calculated from five independent experimental results. Statistical analysis was performed using a tukey test. Values that differ from the control with the 95% confidence level are marked with a star on the top of symbols.

Table 1. Yield of licorice extract according to ethanol content in extract solvent.