Conclusion
These findings demonstrate that the methanolic bark extracts of H. odorata plant induce apoptosis and selective cytotoxicity toward HepG2 but not HF. Therefore, purification of compounds from H. odorata bark extracts may be useful as anticancer agents, and thus, more studies are warranted to investigate the anticancer properties of H. odorata.
Methods
Methanol extracts of H. odorata were prepared from the bark of H. odorata plants (H. odorata extract). The in vitro cytotoxicity of H. odorata extracts on human HCC cell line HepG2 compared to normal human fibroblasts (HFs) was assessed by Alamar Blue assay. Caspase-3/7 was detected using a reagent that consists of DEVD peptide conjugated to a nucleic acid-binding dye. Apoptosis induction by the H. odorata plant extract on HepG2 was evaluated by Annexin V/7-AAD using flow cytometry. Disintegrated nuclei of plant-treated cells were observed under a fluorescent microscope using Hoechst and propidium iodide (PI) staining. In addition, using the Hoechst/PI staining technique, the ratio of dead to total cells was determined by distinguishing Hoechst and PI fluorescent signals.
Results
We found that the IC50 value of H. odorata extract on HepG2 was 12.67±5 µg/mL and on HF was 44±3 µg/mL. The IC50 value of doxorubicin on HepG2 was 153.3±15 ng/mL and on HF was 6.3±0.6 ng/mL. The selectivity index (SI) of H. odorata extract for HepG2 cells was ~3.48, while the SI of doxorubicin for HepG2 cells was ~0.04. The ratio of dead to total cells increased in a dose-dependent manner for HepG2 cells when observed under a fluorescent microscope, while the ratio of dead to total cells barely changed for HF cells. The H. odorata extract inhibited HepG2 cells via the activation of caspase-3/7. At 250 µg/mL concentration of the H. odorata extract, 35% of HepG2 cells were induced into apoptosis, and the cells exhibited disintegrated nuclei under a fluorescent microscope.