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Context: Suspension cultures of Linum album Kotschy ex Boiss. (Linaceae) accumulate podophyllotoxin (an anticancer agent) and could therefore serve as an alternative source of this important aryltetralin lactone lignan. Objective: The present work compared four podophyllotoxin extraction methods and optimization of the best one by using single factor experiments. Materials and methods: Linum album cell cultures were established from in vitro plantlets and subcultured in MS medium with hormones every 7-8 days. Four podophyllotoxin extraction methods were assayed and the best one was optimized by single factor experiments, studying the effect of methanol concentration, extraction time, and sonication time. Results: Cell cultures accumulated enough podophyllotoxin to be analyzed by HPLC. The methanol/dichloromethane and buffer extraction methods were found to be the best. Methanol alone and hot ethanol were not effective for extracting podophyllotoxin. Discussion and conclusion: The optimized method based on methanol/dichloromethane extraction combined with HPLC quantification was able to determine small amounts of podophyllotoxin in Linum album cell cultures, showing that this system could constitute a possible alternative source of podophyllotoxin to Podophyllum (Berberidaceae). [ABSTRACT FROM AUTHOR]