Background: Hepatocellular carcinoma is the most common type of liver cancer and represents a significant global health challenge. Although no universally accepted standard therapy is currently available, the use of natural products in cancer prevention and treatment has gained increasing attention.

Objectives: This study aimed to evaluate the anti-proliferative effects of Panax bipinatifidus Seem. total saponin extract (PBE) on human hepatoma HepG2 cells, with a focus on mitochondrial function, oxidative stress, calcium homeostasis, and cell cycle regulation.

Subjects and Methods: HepG2 cells were treated with various concentrations of PBE for 48 hours. Cell viability, intracellular reactive oxygen species (ROS), cytosolic calcium levels (Ca²⁺), mitochondrial cardiolipin content, and cell cycle distribution were assessed using CCK-8, CM-H₂DCFDA, Fluo-4 AM, NAO staining, and PI-based flow cytometry, respectively. Results: PBE moderately inhibited HepG2 cell proliferation with an IC₅₀ of 33.37 ± 1.30 µg/mL. Treatment with PBE significantly increased intracellular ROS and Ca²⁺ levels compared to control (p <0.05), and markedly reduced mitochondrial cardiolipin content (83.18 ± 4.69% of control, p < 0.05). Moreover, flow cytometry revealed a significant accumulation of cells in the G₁ phase, indicating G₁ cell cycle arrest as a potential mechanism of growth inhibition.

Conclusions: Collectively, these findings suggest that PBE exerts anti-proliferative effects on hepatocellular carcinoma cells by inducing oxidative and calcium stress, disrupting mitochondrial membrane integrity, and arresting the cell cycle at the G₁ phase.