title: 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.
abstract: 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.
authors.affiliation: Bioorganic Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Jammu, Jammu and Kashmir, India.
authors.forename: Imran
authors.initials: I
authors.lastname: Khan
authors.forename: Payare L
authors.initials: PL
authors.lastname: Sangwan
authors.forename: Alamgir A
authors.initials: AA
authors.lastname: Dar
authors.forename: Rather A
authors.initials: RA
authors.lastname: Rafiq
authors.forename: Mufti R
authors.initials: MR
authors.lastname: Farrukh
authors.forename: Jagdish K
authors.initials: JK
authors.lastname: Dhar
authors.forename: Sheikh A
authors.initials: SA
authors.lastname: Tasduq
authors.forename: Surrinder
authors.initials: S
authors.lastname: Koul
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000737:chemistry; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002855:Chromatography, Thin Layer / Q000379:methods*; D006801:Humans; D019684:Magnoliopsida / Q000737:chemistry*; D051379:Mice; D010936:Plant Extracts / Q000737:chemistry* / Q000633:toxicity*; D012878:Skin Neoplasms / Q000188:drug therapy* / Q000503:physiopathology
chemical_list: D015415:Biomarkers; D010936:Plant Extracts
keywords: HPTLC; Platanus orientalis; Quantification; Skin cancer; Triterpenoids