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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 IC 50 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 IC 50 value of ginger root extract of the G3 was 83.3 ± 0.9189 µg/ml, compared to 159 ± 7.6 µg/ml of G1, and 284 ± 5.116 µg/ml of G5 in 2D culture latform; the IC 50 value was 228 ± 33.52µg/ml for G3 compared to 341 ± 3.93 µg/ml for G1, and 603.7 ± 56.33 µg/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.

Anti-tumor activity screening is a typical process used in anti-tumor drug discovery. The ideal anti-tumor drug candidates are extracts or compounds that can inhibit the proliferation of cancer cells via apoptosis, while exerting minimal effects on normal somatic cells. For a long time, fibroblasts were used as normal cells for all anti-tumor screening assays. However, the fibroblasts exhibited several limitations as cell controls for anti-tumor screening. This study aimed to compare the usage of dermal fibroblasts (DFs) and adipose-derived stem cells (ADSCs) as normal cell controls in anti-tumor screening protocols. The DFs and ADSCs were prepared per the published protocols. The IC 50 values of doxorubicin on hepatocellular carcinoma cells HepG2, breast cancer cells MCF-7, DFs and ADSCs were determined via the Alamar blue assay. The side effect indexes (SEIs) were calculated as the ratio of IC 50 values of drugs on cancer cells and IC 50 values of drugs on DFs, and on ADSCs. The stability of the anti-tumor assay was investigated when carried out on DFs and ADSCs from different passages. The results showed that the IC 50 values, as well as SEI values, were not significantly different between using DFs or ADSCs as normal cell controls when DFs and ADSCs were at passage 3. However, for DFs at passage 6 to 12, the IC 50 values of doxorubicin were significantly different between DFs and ADSCs. The IC 50 values of doxorubicin on DFs were strongly reduced due to the senescence of DFs, while the values were more constant in ADSCs. The SEI values of doxorubicin on DFs, compared to HepG2 and MCF-7 cells, were also changed during passage 3 to 12 of the DFs. However, these values were only slightly changed for ADSCs from the 3rd to 12th passages. ADSCs can replace DFs as a normal cell control for anti-tumor activity screening.

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