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The antitumor potential of Gymnosperma glutinosum was previously reported using the in vitro and in vivo L5178Y-R lymphoma murine model. The present study was carried out to isolate and identify the cytotoxic compounds present in the Gymnosperma glutinosum leaf hexane extract. Gymnosperma glutinosum was collected in the semi-arid region of Escobedo, State of Nuevo Leon, Mexico, but it is commonly found in northeastern Mexico; it is traditionally used as a treatment for diarrhea, ulcers and rheumatism. G. glutinosum leaves were extracted with hexane and further fractioned and subfractioned over silica gel by gradient elution with hexane, chloroform, ethyl acetate and methanol. The cytotoxicity of fractions and subfractions was assessed in vitro against L5178Y-R lymphoma cells. Structure elucidation of the active compounds was determined by spectroscopic methods. Fractions and subfractions showed significant (p < 0.05) and concentration-dependent 20% to 56% cytotoxicity against L5178Y-R cells at concentrations ranging from 7.8 mu g/mL to 500 mu g/mL. The bioassay-guided fractionation of the hexane extract resulted in the isolation and identification of the alkane hentriacontane and the diterpene ent-labd-7-en-13S,14R,15-triol as the metabolites responsible for the activity. Copyright (c) 2012 John Wiley & Sons, Ltd.