In vitro chemopreventive activity of an ethyl acetate fraction derived from hot water extract of orthosiphon stamineus in HepG2 cells

Orthosiphon stamineus (Lamiaceae) is a medicinal plant containing several biologically active components that have chemopreventive activity. To investigate the chemopreventive properties of O. stamineus, we studied the apoptotic activity of the ethyl acetate fraction (EAF) derived from the hot water extract of O. stamineus leaves on the human hepatocellular carcinoma cell line, HepG2. The sulforhodomine B assay indicated that the EAF inhibited the viability of HepG2 cells in a concentration dependent manner. Hoechst 33342 staining showed that EAF-treated cells exhibited typical apoptotic morphologic changes such as nuclear condensation and fragmentation. JC-1 assays indicated that the EAF disrupted the mitochondrial transmembrane potential of HepG2 cells in a dose-dependent manner. Western blot analysis revealed that the EAF activated caspase-3, caspase-8 and caspase-9, increased Bax expression, downregulated Bcl-2, decreased Cox-2 expression and decreased level of the NF-kB p65 in nucleus. HPLC-DAD analysis identified the major components in the EAF as rosmarinic acid (31.8%) and caffeic acid (20.2%). Taken together, our study suggests that the EAF has the potential to be developed as an agent for human liver cancer prevention.