Combined Inhibitory Effects of Quercetin and β-Sitosterol on a Hydrogen Sulfide-Producing Enzyme Fn1220 in Fusobacterium nucleatum: A Potential Strategy for Halitosis Control.

AIMS: Fusobacterium nucleatum, an oral opportunistic pathogen, produces hydrogen sulfide (H₂S) from L-cysteine metabolism as a key contributor to halitosis. The enzyme Fn1220 is primarily responsible for H₂S generation in F. nucleatum. This study aimed to screen for natural compounds from Eugenia caryophyllata Thunb. (clove), a traditional herb used for halitosis, that are predicted to interact with Fn1220. METHODS AND RESULTS: Using a computational screening approach (network pharmacology) combined with molecular docking, we identified six components from clove with binding energies below -5 kcal mol⁻¹: strictosamide-qt, quercetin, stigmasterol, β-Sitosterol, kaempferol, and ZINC03860434. Quercetin and β-Sitosterol were selected for further experimental validation. Their effects on H₂S production were quantified via the bismuth chloride colorimetric method, and biofilm formation was assessed by crystal violet staining. The results showed that both compounds, alone or in combination, effectively inhibited F. nucleatum activity in a concentration-dependent manner. At equivalent concentrations, the combination exhibited significantly stronger inhibition than either compound alone. At a total compound concentration of 96 μg/mL (48 μg/mL quercetin plus 48 μg/mL β-Sitosterol), the combination reduced H₂S production by approximately 63% and biofilm formation by approximately 67%. CONCLUSIONS: These findings provide preliminary in vitro evidence that the combined use of quercetin and β-Sitosterol enhances the inhibition of H₂S production and biofilm formation by F. nucleatum, supporting further preclinical investigation.