Effects of Potherb Mustard Extract on Actinomyces naeslundii Biofilm Formation

Objectives: Actinomyces naeslundii is one of initial colonizers on the tooth surface, and is known concerned with periodontal disease and opportunistic infections. Biofilm formation of A. naeslundii was stimulated and up-regulated with short chain fatty acids (SCFAs) produced by oral bacteria. To find safety inhibitors on the biofilm formation, various vegetable extracts were examined. Among these extracts, crude extract from a potherb mustard strongly inhibited the biofilm formation of A. naeslundii stimulated with SCFAs. The purpose of the study is to identify the inhibitory active components in a potherb mustard extract using with antibodies provided by immunizing candidate proteins in a rat.
Methods: Biofilm formation assay of A. naeslundii x600 was performed in TSB with 0.25% (w/v) sucrose containing 6.25 mM butyric acid, one of SCFAs, on the 96 well microtiter plate. To purify the inhibitors, the water extracts of a potherb mustard were performed in DEAE ion-exchange and gel filtration chromatography. Candidate proteins were investigated after screening of inhibiting activities, with SDS-PAGE and TOF-MASS. Candidate proteins were immunized in rats and antibodies were produced. Prepared antibodies were added in the biofilm inhibition assay and their activities were assessed.
Results: As a result of purification of a potherb mustard using DEAE and gel filtration chromatography, the active component was protein and showed high molecule band in native-PAGE. It was also found that six candidate proteins separated with SDS-PAGE were including malate dehydrogenase (MDH) in TOF-MASS. Among six kinds of antibodies produced in rats, No. 2 antibody reacted to 15-40 kDa proteins in SDS-PAGE and Western blotting disturbed the inhibiting activities of extracts in biofilm formation.
Conclusions: These results suggested that 15-40 kDa proteins in a potherb mustard extract, which reacted with No.2 antibody, was containing the inhibitory active components against the biofilm formation of A. naeslundii stimulated with SCFAs.