Optimization of Thymoquinone Extraction from Black Seed Using Natural Deep Eutectic Solvent and Ultrasound-Assisted Extraction

Black seed (Nigella sativa L., Ranunculaceae) possesses antimicrobial activity due to its bioactive compound, thymoquinone. This study aimed to extract thymoquinone from black seed using an alternative natural deep eutectic solvent (NADES) through an optimization approach. A NADES based on malic acid and choline chloride was employed in this work. The Box–Behnken design was used to investigate the effects of the molar ratio of malic acid to choline chloride, water addition, and ultrasonication time. These variables were varied as follows: the malic acid to choline chloride molar ratio ranged from 2:1 to 4:1, the mass ratio of water to NADES from 0.5:2 to 1.5:2, and the ultrasonication time from 60 to 120 min. The results showed that the malic acid to choline chloride molar ratio, the quadratic term of the malic acid to choline chloride molar ratio, and the quadratic term of ultrasonication time significantly affected thymoquinone extraction. The optimal condition, which yielded the highest thymoquinone concentration, was determined to be a malic acid to choline chloride molar ratio of 4:1, a mass ratio of water to NADES of 1:2, and an ultrasonication time of 60 min. Under these conditions, the thymoquinone concentration was 70.57 ± 5.78 µg/mL, closely aligning with the predicted value. The thymoquinone-rich extract obtained using NADES exhibited antimicrobial activity against Staphylococcus aureus, Staphylococcus epidermidis, and Candida albicans. In conclusion, NADES serves as an effective alternative solvent for extracting thymoquinone from black seed.