Evaluation of cytotoxicity of Clinopodium umbrosum on oral cancer cell line HN-5

Abstract

Background: Medicinal plants are especially important in the treatment and prevention of diseases such as cancer. Objective: Due to the prevalence of various cancer types worldwide and the existence of bioactive compounds with different biological activity in C. umbrosum, this species was selected to investigate cell toxicity with the aim of obtaining new cytotoxic compounds. Method: C. umbrosum aerial parts were subejcted to solvent extraction by means of a Soxhlet apparatues. According to the phytochemical results obtained in the previous study, methanolic extract was fractionated by SPE method and then fraction of Sep-pak80% was further fractionated on silica gel using VLC. Subsequently, the two compounds in sub-fraction 7 were isolated by HPLC. Finally, the cytotoxicity activity of the extracts and the two pure compounds on oral cancer cells (HN-5) and Huvec normal cells were investigated by MTT method and flow cytometry. Subsequently, the apoptosis pathway was studied through Real-time PCR. Besides, scratch test was performed to study the cell migration. Results: The cytotoxic activity of petroleum ether, chloroform and methanol extracts on HN-5 oral cancer cells after 24 hours of treatment with IC50 were >0.25, >0.167 and 0.24mg/mL, respectively. After 48 hours the IC50 results were >0.25, >0.167 and 0.23mg/mL, respectively. The cytotoxic findings for the two compounds Buddlejasaponin IVa and Buddlejasaponin IV showed that Buddlejasaponin IV has more cytotoxicity and both compounds showed their cytotoxicity through apoptotic pathway with increasing Bax/Bcl2 ratio and the level of caspase 9. Also HN-5 cell migration was reduced with two saponin compounds. Conclusion: Methanol extract of C. umbrosum possessed significant cytotoxicity on HN-5 cells and the mechanism of cytotoxicity of its two major compounds, Buddlejasaponin IVa and Buddlejasaponin IV, was identified as mitochondrial pathway of apoptosis reducing the invasive potential of HN-5 cells.