Anticancer activity of ionic liquid graviola fruit extract on MCF-7 and HT29 cancer cell lines using in vitro and in vivo methods

Abstract

Cancer, one of the major public health problems, is the leading cause of death worldwide. The available protocols of treatment include surgical intervention, radiation, and chemotherapy which cause numerous side effects on cancer patients. Many phytochemicals have anticancer properties comparable to conventional drugs. The major benefit of these compounds is the non-toxicity nature to the normal tissues. Annona muricate, commonly known as Graviola, is a member of the Annonaceae family. It has been used for ages in traditional medicine due to its biological activities including antioxidant, anti-inflammatory, antimicrobial and cytotoxicity to tumour cells. This research investigated the antiproliferative effect of the ionic liquid-Graviola fruit extract (IL-GFE) on in vitro breast MCF-7 and colon HT29 adenocarcinoma cell lines and their cytokinetic behaviour. It also identified the mechanism of IL-GFE inhibition by applying a flow cytometry technique and metabolomics study and assessed its toxicity on in vivo zebrafish developing embryos. The process of ionic liquid-microwave assisted extraction (IL-MAE) method was optimised by Response Surface Methodology for three parameters, namely time, irradiation power and solid-liquid ratio. The optimum extraction conditions gave a yield of Graviola fruit extract up to 66.6 % and an average IC50 of 4.75 µg/mL for MCF-7 and 10.56 µg/mL for HT29, while it was safe toward normal VERO cell lines. The crude IL-GFE was fractionated using the combination of thin-layer chromatography and column chromatography. Six fractions were semi-purified and subjected to phytochemical screening and antiproliferative assay in which, it revealed the presence of many phytoconstituents such as acetogenins, alkaloids, phenols, flavonoids, tannins and terpenoids. Moreover, fraction B exhibited the lowest IC50 toward MCF-7 and HT29 cells at 12.6 and 13.56 μg/mL, respectively. However, the crude IL-GFE had a better IC50 value. The crude IL-GFE with GC-TOFMS analysis revealed the presence of many phytochemicals with anticancer activity such as D-psicofuranose, pentakis ether, propyldecyl cyclopropane carboxylate, tri-ruthenium dodecacarbonyl, N-acetylimino dimethylsulfurane, pyranone, carbohydrazide and benzoic acid. The cytokinetic study showed that crude IL-GFE and Taxol inhibited the growth of MCF-7 and HT29 cells and proved their antiproliferative effect when they reduced the number of cell generations of MCF-7 from 3.71 to 1.67 and 2.18, respectively, and reduced the cell generations of HT29 cells from 3.93 to 2.96 and 2.01, respectively. Furthermore, the acute toxicity of IL-GFE was assessed on in vivo zebrafish model in which crude IL-GFE reduced the survival of zebrafish larvae at a relatively high dose of 250 µg/mL after 96 hpf treatment, while no significant changes on morphology of the treated zebrafish were recorded. The result of the flow cytometry also indicated that the crude IL-GFE arrested the cell cycle of MCF-7 and HT29 at G0/G1 phase and increased the apoptotic and necrotic cells in a time-dependent manner compared with the control group. Finally, the metabolomics analysis of the treated MCF-7 and HT29 cells with crude IL-GFE treatment showed an alteration of many metabolic pathways in treated cancer cells. In conclusion, crude IL-GFE can be one of the promising anticancer agents due to its selective antiproliferation against breast and colon cancer cells and its safety for the healthy cells.