Jin-ling Zhou,1,* Xiu-ying Huang,2,* Han-chen Qiu,3 Ri-zhi Gan,1 Huan Zhou,1 Hong-qing Zhu,1 Xuan-xuan Zhang,1 Guo-dong Lu,4 Gang Liang1 1School of Pharmacy, Guangxi Medical University, Nanning, People’s Republic of China; 2Liuzhou Employment Service Centre for the Disabled, Liuzhou, People’s Republic of China; 3Department of Pharmacy, The People’s Hospital of Hezhou, Hezhou, People’s Republic of China; 4School of Public Health, Guangxi Medical University, Nanning, People’s Republic of China*These authors contributed equally to this workCorrespondence: Gang LiangDepartment of Pharmacology, School of Pharmacy, Guangxi Medical University, Nanning 530021, Guangxi Province, People’s Republic of ChinaTel + 86 771 5302271Fax + 86 771 5358272Email lianggang@gxmu.edu.cnGuo-dong LuDepartment of Toxicology, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi Province, People’s Republic of ChinaTel + 86 771 535 8114Fax + 86 771 5350823Email golden_lu@hotmail.comIntroduction: Saponin of Schizocapsa plantaginea Hance I (SSPH I), a novel bioactive phytochemical isolated from the rhizomes of Schizocapsa plantaginea, has been demonstrated to exhibit anti-cancer activity against various tumors in preclinical studies. However, the molecular mechanisms involved in the suppression of hepatocellular carcinoma (HCC) are poorly understood. The present study aimed at analyzing the effects of SSPH I on autophagy and apoptosis in vitro.Methods: MTT and colony forming assays were used to detect cell viability and cell proliferation. Hoechst 33,258 staining and flow cytometry were used to determine apoptosis and ROS production. The apoptosis and autophagy-related protein expression levels were evaluated via Western blot assay. Characteristics of autophagy and apoptosis were observed by transmission electron microscopy. Lysosomal activity was stained with Lyso-Tracker Red and Magic Red Cathepsin B.Results: The results showed that SSPH I exhibited potent anti-cancer activity and proliferation in HepG2 and BEL-7402 cells and inhibited HepG2 cells through inhibiting autophagy and promoting apoptosis. The mechanistic study indicated that the inhibition of autophagy of SSPH I was mediated by blocking autophagosome–lysosome fusion. Additionally, we found that SSPH I could mediate the activation of MAPK/ERK1/2 signaling pathway, and the use of NAC (ROS inhibitor) and U0126 (MEK1/2 inhibitor) converted the effect of SSPH I on apoptosis and autophagy in HepG2 cells.Conclusion: These data suggest that SSPH I induces tumor cells apoptosis and reduces autophagy in vitro by inducing ROS and activating MAPK/ERK1/2 signaling pathway, indicating that SSPH I might be a novel agent for the treatment of HCC.Keywords: saponins of Schizocapsa plantaginea Hance I, SSPH I, autophagy, hepatocellular carcinoma cells, apoptosis, ROS, MAPK