Abstract
INTRODUCTION: Physochlaina physaloides (L.) G. Don is a traditionally used botanical drug whose antitumor activity has been linked to flavonoid-enriched fractions. This study aimed to optimize the ultrasound-assisted extraction (UAE) of total flavonoids from the roots of P. physaloides and to evaluate the cytotoxic activity of the resulting optimized flavonoid extract (OFE) in human cervical cancer cells. METHODS: Total flavonoids were prepared from the roots of P. physaloides using UAE. A Box-Behnken response-surface design was employed to optimize the extraction conditions. The effects of the solid-liquid ratio, extraction temperature, and ultrasonic power on flavonoid yield were assessed. The bioactivity of OFE was then evaluated in HeLa cells using the MTT assay, Calcein-AM/PI staining, Annexin V-FITC/PI apoptosis analysis, and western blotting of apoptosis- and survival-associated proteins. RESULTS: The solid-liquid ratio, temperature, and ultrasonic power were identified as the key factors influencing flavonoid yield. The optimized extraction conditions were 80% ethanol, a solid-liquid ratio of 1:60 g/mL, 50 °C, 335 W, and 40 min, yielding 11.005 ± 0.663 mg/g of total flavonoids, which closely matched the model prediction. In HeLa cells, OFE reduced cell viability in a dose- and time-dependent manner, with the most pronounced effect observed at 48 h in both the MTT assay and Calcein-AM/PI staining. Annexin V-FITC/PI analysis showed that apoptotic cell populations increased with escalating OFE exposure. Mechanistically, OFE treatment was associated with reduced p-PI3K and p-AKT levels and increased cleaved caspase-3 expression. DISCUSSION: These findings establish a practical UAE procedure for the preparation of total flavonoids from P. physaloides and provide in vitro evidence that OFE exerts cytotoxic effects in HeLa cells by promoting apoptosis-associated responses, potentially through modulation of PI3K/AKT-related survival signaling.