Zohre Ghanavi - Department of Horticultural Science and Agronomy, Science and Research Branch, Islamic Azad University, Tehran, Iran
Ali Akbar Velayati - Mycobacteriology Research Center (MRC), National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran
Parissa Farnia - Mycobacteriology Research Center (MRC), National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran
Amir Naji - Department of Agronomy and Crop Breeding Faculty of Agriculture, Shahed University, Tehran, Iran
Sepideh Kalatehjari - Department of Horticultural Science and Agronomy, Science and Research Branch, Islamic Azad University, Tehran, Iran

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 C۱۸ column and the mobile phase of methanol and water containing ۰.۱% 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 ۹۷%, while the results of GC-MS identified the main peak appeared at around ۱۵.۶ min as TQ by library searching, and determined its purity by ۹۷% based on peak heights. Overall, this method has a potential for industrialization to prepare purified TQ for medicinal applications.

Nigella sativa L, Thymoquinone, PURIFICATION, Preparative HPLC