The Antitumor Mechanism of the Polyphenol-enriched Ethyl Acetate Fraction Extract of Glechoma hederacea (Lamiaceae) against HepG2 Cells Involves Apoptosis Pathways

Abstract Background: Glechoma hederacea (Lamiaceae) is a traditional Chinese herb belonging to the Labiatae family and has many biological activities. This study aimed to investigate the in vitro effects of different polar parts of the water extracts of G. hederacea (Lamiaceae) on HepG2 cells and the possible underlying mechanism.Methods: The cytotoxic effects were determined using the MTT assay. Apoptosis was quantified using annexin V-FITC/PI staining. Caspase-3 and caspase-9 activities were measured using PhiphiLux-G1D2 kit. Apoptosis-related protein expression was determined using western blot analysis. Reactive oxygen species (ROS) generation, mitochondrial transmembrane potential (MMP), and calcium ion levels were measured using specific fluorescence probes and flow cytometry. The HepG2 cell antioxidant state was determined using DAPI and comet assay, and antioxidant enzyme activities. The polyphenol contents of different polar parts of fractional extracts were determined using HPLC. Results: Our results showed that rosmarinic acid, caffeic acid, and ferulic acid were the major polyphenolic components in the ethyl acetate fraction extract (EAFE) of G. hederacea L.. EAFE could significantly inhibit HepG2 cell proliferation, induce apoptosis, and cause S phase arrest. The apoptogenic activity of EAFE involved ROS induction, Ca2+ accumulation, glutathione depletion, MMP disruption, caspases 3, 9 activation, and Bax/Bcl-2 ratio increment.Conclusion: Our findings suggest that the polyphenols in the EAFE can inhibit HepG2 cell growth and induce apoptosis through the mitochondria-mediated pathway and ROS-mediated endoplasmic reticulum stress. EAFE could be developed as a functional food or nutraceutical ingredient for chemotherapy.