Betulinic acid

TLC analysis was carried out using activated silica gel 60 F₂₅₄ TLC plates (Merck, Germany) as the stationary phase. Betulinic acid (Chemfaces, China) and lupeol (Chemfaces, China) were utilized as standard references. Five microliters of each 1 mg/mL standard reference solution and each 15 mg/mL sample extract were loaded with a 4 mm bandwidth on TLC plates using a sample applicator (Linomat V, Camag, Switzerland) equipped with a 100 μL syringe. Then the plates were developed in the optimized mobile phase to a distance of 80 mm. After that, the dried plates were sprayed with anisaldehyde-sulfuric acid reagent before drying in an oven at 105°C for 20 min. The TLC chromatograms were viewed and photo-recorded under UV light at 366 nm using a TLC viewer (TLC visualizer 2, Camag, Switzerland). The results from this method were orderly rearranged using visionCATS version 2.3 software.

PLGA (Mol. wt. 76.05), PVA (Mol wt. 89,000–98,000), quercetin, chlorhexidine and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide), DAB (Diaminobenzidine) and RPMI 1640 were purchased from Sigma Aldrich Ltd. (St. Louis, MI, USA). Fetal bovine serum (FBS) was purchased from Biosera (Kansas City, MO, USA). Trypan blue, Phosphate buffer saline (PBS), Dichloromethane (DCM), Ethanol, Paraformaldehyde and Dimethyl sulfoxide (DMSO) were purchased from IDL Scientific Ltd. (Kuala Lumpur, Malaysia). Periglaucine A, betulinic acid, dichloromethane ChemFaces Ltd. (Hubei, China). In situ apoptosis detection kit was purchased from Millipore, Merck (MA, USA).