Among oral diseases, dental caries is one of the most frequent to affect human health. The current research work aimed to ascertain the antibacterial, anti-biofilm, and antioxidative potential of Piper betle leaf extract against bacteria isolated from dental caries. Analysis for the presence of phytochemical compounds revealed compounds, such as tannins, steroids, phenolic compounds, and alkaloids, which were also confirmed by TLC and FTIR. GC-MS analysis elucidated the presence of 20 phytocompounds, among which were some well-reported bioactive compounds. The chloroform extract of P. betle demonstrated good antibacterial activity (7 mm) and minimum inhibitory concentration (MIC) (100 mg mL-1) against Bacillus gaemokensis MW067143, which was the frequent biofilm producer among isolated bacterial strains. Fractions of the extract were isolated through column chromatography, after which the antibacterial activity was again evaluated. Spirost-8-en-11-one,3-hydroxy(3β,5α,14β,20β,22β,25R), an oxosteroid in nature, was observed to exhibit remarkable antibacterial potential (12 mm) against B. gaemokensis. Bacterial cells treated with P. betle extract had elevated SOD, APOX, POX, and GR activity, while its proteolytic activity against whole bacterial proteins was pronounced with the suppression of several proteins (50, 40, 15, and 10 kDa) in SDS-PAGE. Bacterial cells treated with P. betle extract demonstrated decreased growth, while the extract was also observed to exhibit inhibition of biofilm formation (70.11%) and demolition of established B. gaemokensis biofilms (57.98%). SEM analysis revealed significant changes to bacterial morphology post treatment with P. betle, with cellular disintegration being prominent. In silico network pharmacology analysis elucidated proteins like ESR1 and IL6 to be majorly involved in biological pathways of dental caries, which also interact with the protective ability of P. betle. Gene Ontology (GO) terms and KEGG pathways were also screened using enrichment analysis. Molecular docking demonstrated the highest binding affinity of Spirost-8-en-11-one,3-hydroxy-,(3β,5α,14β,20β,22β,25R) with bacterial proteins FabI (-12 kcal/mol), MurB (-17.1 kcal/mol), and FtsZ (-14.9 kcal/mol). Therefore, it is suggested that P. betle can serve a potentially therapeutic role and could be used in the preparation of herbal formulations for managing bacterial flora.
Keywords: Bacillus; Piper betle; antibacterial activity; dental caries; molecular docking; scanning electron microscopy.