A validated high-performance thin-layer chromatography method for the identification and simultaneous quantification of six markers from Platanus orientalis and their cytotoxic profiles against skin cancer cell lines.

Betulinic acid (1), betulinic acid-3-acetate (2), 3-acetylbetulinaldehyde (3), oleanolic acid-3-acetate (4), 3-β-hydroxy-28,19-β-olenolide (5), and β-sitosterol (6) were isolated from Platanus orientalis and a high-performance thin-layer chromatography method was developed for their simultaneous quantification. The markers were first derivatized on the chromatogram with ceric ammonium sulfate and then high-performance thin-layer chromatography densitometry was carried out. Chromatographic separation of these markers was carried out on silica gel 60 plates using a ternary solvent system n-hexane/toluene/acetone (6:3.5:1 v/v/v) as a mobile phase. For marker 1, a deuterium (D2) lamp and wavelength of 420 nm was used. A tungsten (W) lamp was used for markers 2 and 3 at 550 nm and for 4-6 at 500 nm. The method was validated for accuracy, precision, LOD, and LOQ. All calibration curves showed a good linear relationship (r > 0.9919). The precision evaluated by an intra- and interday study showed RSDs < 2.51% and accuracy validation recovery between 95.54 and 99.33% with RSDs < 1.55%. The successful application of the validated method showed 1 as the most abundant component (4.63%) and 5 (0.017%) the least. The markers displayed a significant cytotoxic effect against human keratinocyte, mouse melanoma, and human skin epithelial carcinoma cancer cells by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.