Induction of apoptosis in HepG2 by Vitex agnus-castus L. leaves extracts and identification of their active chemical constituents by LC-ESI-MS

Abstract

Objective To evaluate the cytotoxic activity and cytopathological changes of Vitex agnus-castus L. ( V. agnus-castus ) leaves extracts and characterize their bioactive chemical constituents. Methods The dried leaves powder of V. agnus-castus was extracted using 85% methanol (MeOH). The methanolic extract was defatted using petroleum ether and fractionated using ethyl acetate (EtOAc) and butanol (BuOH). The anticancer potential of different extracts was evaluated by neutral red assay, cytopathological changes of apoptosis and caspase-3 expression in hepatoma cell line (HepG2). The chemical constituents of most active extracts were identified using liquid chromatography-electrospray ionisation mass spectrometry analysis. Results The butanolic fraction was the most active in inhibiting the proliferation of HepG2 cells [IC 50 = (13.42 ± 0.17) mg/mL] compared with MeOH extract [IC 50 = (17.61 ± 0.15) mg/mL) and EtOAc fraction [IC 50 = (22.51 ± 0.26) mg/mL]. The cytopathological examinations demonstrated the morphology of apoptosis and caspase-3 expression was more evident in HepG2 cells treated with BuOH than cells treated with MeOH and EtOAc. The liquid chromatography-electrospray ionisation mass spectrometry analysis exhibited that the defatted MeOH extract and BuOH fraction had different bioactive secondary metabolites, such as phenolic acids, flavonoids, and iridoids. Conclusions The butanolic fraction has higher contents of secondary metabolites than the defatted methanolic extract. The cytotoxic activities, apoptotic changes, and caspase-3 activation may be due to the presence of these bioactive secondary metabolites (iridoids, flavonoid, and phenolic acids) in these extracts. These results would suggest V. agnus-castus to be used as an adjuvant in cancer therapy.