Optimization of Different Methods for the Extraction of Mulberry Leaves and the Effects on Caco-2 Cells

Mulberry (Morus nigra Aussie) leaves have traditionally been used in silkworm rearing and as herbal remedies, yet their extracts have recently gained prominence in functional foods due to their rich phenolic composition and associated health benefits, including antioxidant, hypoglycemic, anti-obesity, anti-inflammatory, and potential anticancer effects. Because extraction techniques strongly influence phenolic yields, this study optimized the extraction conditions for conventional, ultrasonic-assisted, and microwave-assisted methods using response surface methodology (RSM). Three independent factors—extraction temperature/power, extraction time, and solvent concentration (ethanol–water mixture, % v/v)—were evaluated for their effects on total phenolic content. The optimum conditions were identified as 60 °C, 60 min, and 56% ethanol for the conventional method; 60 °C, 45 min, and 71% ethanol for the ultrasonic-assisted method; and 471 W, 6 min, and 51% ethanol for the microwave-assisted method. At these optima, the total phenolic contents (TPCs) were 876 mg/kg (conventional), 820 mg/kg (ultrasonic-assisted), and 319 mg/kg (microwave-assisted) on a dry-leaf basis. Ultrasonic-assisted extraction produced phenolic yields comparable to those obtained by the conventional method. Therefore, its potential anticancer activity was assessed using Caco-2 cells. However, the extract inhibited cell viability of only 12% after 24 h, indicating no significant anticancer effect (cell viability remained >70%). These findings highlight optimized green extraction conditions for mulberry leaves while demonstrating that the ultrasonic-assisted extract lacks anticancer activity under the tested conditions.