Petroleum ether

The three anticancer biomarkers, ursolic acid, β-sitosterol and lupeol were procured from Sigma Aldrich (USA). The solvents used (petroleum ether, ethyl acetate, toluene, chloroform, ethyl acetate, methanol and n-butanol; AR grade) were purchased from BDH (UK). The HPTLC plates (glass-backed silica gel 60F₂₅₄) were purchased from Merck (Germany). The application of biomarkers and extracts (band wise) to the plate was carried out by Automatic TLC Sampler-4 (CAMAG, Switzerland) while the development took place in automatic development chamber (ADC2, Switzerland). The developed plate was derivatized with p-anisaldehyde reagent [the reagent was prepared according to the Wagner and Bladt (2004) by using anisaldehyde, glacial acetic acid, methanol and concentrated sulphuric acid. Initially anisaldehyde (0.5 mL) was mixed with glacial acetic acid (10 mL) followed by addition of methanol (85 mL) and then sulphuric acid (5 mL)]. The scanning and documentation of developed HPTLC plate was carried out by CATS 4 (CAMAG, Switzerland) and TLC Reprostar 3 (CAMAG, Switzerland), respectively.