Extraction and Purification of Anticancer Thymoquinone from Seeds of Nigella sativa by Preparative High-performance Liquid Chromatography

Thymoquinone (TQ) is a monoterpene ketone which is an important bioactive substance in Nigella sativa L. seed. This important natural compound has many potential medical applications for cardiovascular diseases, diabetes, stroke, neurodegenerative diseases and different types of cancer. To date, no studies have focused on development of a reliable industrial method for purification of TQ from this plant as a major challenge. In this study, an efficient extraction and purification method was developed for preparation of TQ from Nigella sativa L. seeds using preparative high-performance liquid chromatography (HPLC). To this end, a two-step procedure was applied for the first time for extraction of TQ: first, maceration using methyl tert-butyl ether (MTBE) was performed; then, liquid-liquid extraction using methanol successfully removed the majority of the impurities. Next, preparative HPLC was carried out for separation and purification of TQ using a C18 column and the mobile phase of methanol and water containing 0.1% trifluoroacetic acid (TFA).The collected peak from preparative HPLC was analyzed by the analytical HPLC as well as GC-MS instrument. Results of HPLC analysis proved the purity of the collected TQ by 97%, while the results of GC-MS identified the main peak appeared at around 15.6 min as TQ by library searching, and determined its purity by 97% based on peak heights. Overall, this method has a potential for industrialization to prepare purified TQ for medicinal applications.