Ultrasound-Assisted Extraction of Cytotoxic Alkaloids from Caliphruria subedentata: Comparative Analysis of Bath and High-Intensity Systems

Cancer remains one of the leading causes of death worldwide, and despite significant therapeutic advances, the discovery of effective and sustainable chemotherapeutic agents remains a major challenge. Alkaloids from the Amaryllidaceae family are well known for their cytotoxic potential, yet their complex phytochemical matrices and low alkaloid contents often lead to low extraction yields and high solvent consumption. This study aimed to optimize the extraction of cytotoxic Amaryllidaceae alkaloids from Caliphruria subedentata using unconventional ultrasound-assisted extraction techniques and to evaluate their activity against gastric cancer (AGS) cells. Three extraction approaches were compared: Soxhlet extraction (conventional), bath ultrasound-assisted extraction (BUAE), and high-intensity ultrasound-assisted extraction (HIUAE). Alkaloid profiles were assessed by gas chromatography-mass spectrometry (GC–MS), and cytotoxicity was assessed using the MTT-based colorimetric method. A two-factor, two-level experimental design combined with response surface analysis was applied for optimization. HIUAE provided the highest alkaloid yield and cytotoxic activity, with an inhibitory concentration corresponding to 50% of the maximal effect (IC₅₀) values of 0.42 ± 0.23 µg/ml, 10.30 ± 3.76 µg/ml, and 70.34 ± 3.08 µg/ml for HIUAE, BUAE, and Soxhlet, respectively. Optimal extraction conditions were a methanol: water ratio of 8.28:1.72, a solid-to-liquid ratio of 1:10, and an extraction time of 40 s. The optimized extract exhibited enhanced cytotoxicity consistent with its higher alkaloid content. Overall, HIUAE represents an efficient, rapid, and environmentally friendly method for obtaining bioactive alkaloids from C. subedentata , supporting the development of green extraction strategies for potential anticancer agents.