In vitro anti–leukemia activity of Gynostemma pentaphyllum (Thunb.) on HL-60 leukemia cell line

Objective: This study aimed to investigate the in vitro anti-leukemia activities of the active extract derived from the leaves of Gynostemma pentaphyllum, focusing on its cytotoxic properties, cell cycle regulation, and impact on key protein markers involved in leukemia cell proliferation. Materials and methods: G. pentaphyllum fractional extracts were prepared through sequential extraction using hexane, ethyl acetate, and ethanol solvents through maceration. The cytotoxicity of these extracts was determined against HL-60 leukemia cell line using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The most cytotoxic extract was further fractionated through column chromatography. The anti-leukemia activities of the active extract and its active fraction were evaluated through cell cycle analysis using flow cytometry. Western blotting was performed to assess the expression levels of key proliferation markers, Feline McDonough Sarcoma (FMS)-like tyrosine kinase 3 (FLT3) and Wilms’ tumor 1 (WT1) proteins. Results: The intermediate-polar ethyl acetate fractional extract (F-EtOAc) was found to be highly cytotoxic against HL-60 leukemia cells, with an IC50 value of 43.41±2.75 μg/mL. Among the isolated fractions of F-EtOAc, fraction F10 was identified as the most active. It was shown that F-EtOAc and F10 significantly induced S-phase cell cycle arrest and reduced the expression levels of FLT3 and WT1 proteins in HL-60 cells in a dose-dependent manner (p <0.001), suggesting that anti-proliferative molecular pathways contribute to their anti-leukemia potential. Conclusion: This study’s findings indicate that the ethyl acetate extract of G. pentaphyllum contains bioactive compounds with anti-leukemia potential, supporting further investigation into its underlying molecular mechanisms.