Supplementary Material for: In vitro Antimicrobial Activities of 1-Methoxyficifolinol, Licorisoflavan A, and 6,8-Diprenylgenistein against Streptococcus mutans

The objective of the study was to investigate the antimicrobial
effects of purified single compounds from ethanol-extracted licorice
root on Streptococcus mutans. The crude licorice root extract (CLE) was obtained from Glycyrrhiza uralensis,
which was subjected to column chromatography to separate compounds.
Purified compounds were identified by mass spectrometry and nuclear
magnetic resonance. Antimicrobial activities of purified compounds from
CLE were evaluated by determining the minimum inhibitory concentration
and by performing time-kill kinetics. The inhibitory effects of the
compounds on biofilm development were evaluated using crystal violet
assay and confocal microscopy. Cell toxicity of substances to normal
human gingival fibroblast (NHGF) cells was tested using a methyl
thiazolyl tetrazolium assay. Chlorhexidine digluconate (CHX) was used in
the control group. Three antimicrobial flavonoids,
1-methoxyficifolinol, licorisoflavan A, and 6,8-diprenylgenistein, were
isolated from the CLE. We found that the three flavonoids and CHX had
bactericidal effects on S. mutans UA159 at the concentration of ≥4 and ≥1 µg/ml, respectively. The purified compounds completely inhibited biofilm development of S. mutans
UA159 at concentrations over 4 μg/ml, which was equivalent to 2 μg/ml
of CHX. Confocal analysis showed that biofilms were sparsely scattered
in the presence of over 4 μg/ml of the purified compounds. However, the
three compounds purified from CLE showed less cytotoxic effects on NHGF
cells than CHX at these biofilm-inhibitory concentrations. Our results
suggest that purified flavonoids from CLE can be useful in developing
oral hygiene products, such as gargling solutions and dentifrices for
preventing dental caries.