Ethanol extract of Ginger Zingiber officinale Roscoe by Soxhlet method induces apoptosis in human hepatocellular carcinoma cell line

Abstract

Introduction: Ginger has had a long history of use in healthcare in Asian countries, including Vietnam. Internationally, there have been a few studies done to assess the anticancer activity and potency of ginger. In this study, we investigated the activity (cytotoxicity) of various ginger root extracts that were produced by different preparation methods on human hepatocellular carcinoma cell line (HepG2 cell line).

Methods: Ginger (Zingiber officinale Roscoe) root extract were extracted from the fresh ginger roots by three methods, which included maceration (G1), Soxhlet (G3), and ultrasonic (G5) methods. The extracts were then evaluated for cytotoxic activity on HepG2 hepatocellular carcinoma cell line based on IC₅₀ calculation. The most potent extract was then evaluated for its cytotoxicity by Propidium iodide (PI) staining method and microscopic examination by fluorescence microscopy for both 2D and 3D culture conditions. After treatment, cells were also stained with Annexin-V to assess the apoptosis-inducing ability of the extract.

Results: The results showed that the ginger root extract prepared by the Soxhlet method (G3) had the strongest activity, and showed significant differences compared to extracts from methods G1 and G5, on the hepatocellular carcinoma cell lines cultured in both 2D and 3D conditions. Indeed, the IC₅₀ value of ginger root extract of the G3 was 83.3 +/- 0.9189 ug/ml, compared to 159 +/- 7.6 ug/ml of G1, and 284 +/- 5.116 ug/ml of G5 in 2D culture latform; the IC₅₀ value was 228 +/- 33.52 ug/ml for G3 compared to 341+/- 3.93 ug/ml for G1, and 603.7 +/- 56.33 ug/ml for G5 in 3D culture platform. The results of PI staining of cells in 2D and 3D culture conditions showed that ginger root extract killed HepG2 cells in a concentration-dependent manner. The nuclei of HepG2 cells in 2D culture showed nuclear fragmentation when treated with ginger root extract; this is one of the apoptosis indicators. Moreover, Annexin-V staining results showed that HepG2 cells underwent apoptosis when treated with ginger root extract.

Conclusion: The results of the study show that ginger root extract prepared by the Soxhlet method induced strong cytotoxicity of hepatocellular carcinoma cells cultured in both 2D and 3D conditions, through induction of apoptosis. The evidence suggests that ginger is a potential agent for anticancer therapy. It is necessary to perform further research to isolate compounds from fresh ginger root extract by the Soxhlet method to evaluate the mechanism of anti-tumor activity.