Prestoblue

The leaves of the plants considered for the current study were gathered at the Hatia region of Ranchi district, Jharkhand, India. The plants were then identified at the Department of Botany, St. Xavier's College, Ranchi, Jharkhand, India, by Dr. Ajay Srivastava (HOD, Department of Botany, St. Xavierme College, Ranchi, Jharkhand, India). A herbarium specimen voucher for each plant was kept at the herbarium of the department of Botany at St. Xavier's College.
DPPH and PrestoBlue^® were procured from Sigma Aldrich, South Africa. Brain–heart infusion (BHI) broth and agar, Anaerocult^®, and McFarland standard 1 were purchased from Merck Chemicals (Pty) Ltd. (Wadeville, South Africa). Chlorhexidine (CHX) gluconate was bought from Dental Warehouse (Sandton, South Africa) and S. mutans were bought from the ATCC 25175.

Cells (3*10⁴) were cultured in 24-well plates under standard conditions (5% CO₂, 95% humidity at 37 °C). After overnight incubation, the medium was removed and metabolic cell activity was measured with PrestoBlue® (Invitrogen, Karlsbad, CA, USA) in triplicate according to the manufacturer's instructions. Afterwards, the cells were starved in the medium with only 0.1% FBS and without P/S and supplements (starving medium) for 1 h and the transwells with the K-wires were added.
After three days of cultivation, the PrestoBlue assay was followed by an alkaline phosphatase (AP) activity assay using 0.13% w/v 4-nitrophenyl phosphate disodium salt hexahydrate (p-NPP, Sigma-Aldrich/Merck, Darmstadt, Germany) dissolved in 0.1 M AP-buffer pH 10.5 (50 mM glycine, 100 mM Tris-Base, 1 mM MgCl₂ in dH₂O). Cells were incubated for 30 min at 37 °C and absorbance was read in triplicate at a wavelength of 405 nm.