The n-hexane extract from spearmint (Mentha spicata) leaves was purified via a bioassay-guided fractionation scheme, which was based on the induction of differentiation in a RCM-1 human colon cancer cell line. One of the criteria of a differentiation marker is the formation of ducts determined visually using phase contrast microscopy, the most potent fraction contained piperitenone oxide, based on EI-MS, H-1-NMR and C-13-NMR spectra. Previously, the role of piperitenone oxide was considered as an odor producing compound in spearmint cultivars, but this study indicates the potential medical benefits of this compound. The effective dose of piperitenone oxide was lower than that of any differentiation-inducers found in the bioassay. Piperitenone oxide was categorized into monoterpene such as carvone and menthol, which are also familiar ingredients in Mentha genus plants. However, carvone and menthol did not possess the differentiation-inducing effect. (C) 2014 Elsevier Ltd. All rights reserved.