en Cancer Cancer Original Article Original Article Abstract Our previous studies showed that various extracts of Persian shallot (Allium hirtifolium) have anti-angiogenic effects. This study has been undertaken to isolate and identify the major effective anti-angiogeneic sub-fraction of shallot. After preparation of the 50% hydroalcoholic extract of shallot bulbs, the extract was successively fractionated into n-hexane, ethyl acetate, n-butanol and aqueous fractions. Anti-angiogenesis activity of fractions was examined by in vitro angiogenesis assay. The ethyl acetate fraction which had the most anti-angiogenesis activity was further fractionated to four sub-fractions by thin layer chromatography (TLC), silica gel column chromatography and then analyzed by High Performance TLC (HPTLC) with ethyl acetate-methanol-water as the solvent system. Our results showed that one of the four sub-fractions, as the major band in HPTLC, had the most anti-angiogenic activity. Purification and characterization of the major anti-angiogenic compound/compounds of shallot's extract may constitute one means by which diets rich in shallot confer protection against cancer and finally introduce new agents with pharmacological activities in shallot as a potential candidate in cancer therapy. Allium hirtifolium, Angiogenesis, Phytochemicals, Cancer, High performance thin layer chromatography 237 245 http://ijmcmed.org/browse.php?a_code=A-10-260-1&slc_lang=en&sid=1 Shima Famil Samavati sh.famil.s@gmail.com 10031947532846004122 10031947532846004122 No Department of Biology, Islamic Azad University, Science and Research Branch, Tehran, Iran. Hamid-Reza Mohammadi-Motlagh mohammadimotlagh@gmail.com 10031947532846004123 10031947532846004123 Yes Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran. Ali Mostafaie amostafaie@kums.ac.ir 10031947532846004124 10031947532846004124 No Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran.