Phytochemical analysis of Eleutherine americana MERR ethanol extract and its anticancer properties against breast cancer spheroids

Prevalent among women, breast cancer is the most common form of cancer affecting women in Malaysia; about one in nineteen women in this country are at risk. Bioactive compounds extracted from plants (phytotherapy) have long been recognized as one of the sources of anticancer drugs, several of which have been validated to have potent anticancer properties such as Taxol (paclitaxel). Eleutherine americana (E. americana) possesses a number of phytochemical compounds with the potential to be developed as an anticancer drug. The objectives of this study were to evaluate the phytochemical properties of E. americana ethanol extract, to assess its cytotoxicity on breast cancer cells and to determine its anti-cancer properties on the spheroids and its cancer-associated fibroblasts. In the phytochemical qualitative analysis, E. americana’s bulb extract demonstrated the presence of terpenoids, flavonoids, glycosides and steroids known as beneficial pharmacology bioactive compounds. In the quantitative analysis, the bulb extract displayed significant results on the total phenolics at 151.13 ± 0.027 mg GAE/g, total flavonoids at 22.02 ± 0.005 QE/g and total alkaloids at 2.26 mg ± 0.381/50 g. The extract also showed antioxidant properties with the DPPH assay results measured at 47.60 µg/mL. These results provide validation that the plant extract used in this study contains beneficial pharmacological properties. The invasion assay showed a significant reduction in the invasion area in both monoculture and co￾culture treated with E. americana extract, indicating the anti￾proliferative activity of the plant extract. In addition, the H&E staining of MDA-MB-231 spheroids treated with E. americana ethanol extract showed extensive damage to the nucleus, indicating cell death. This study applies the use of cell spheroids which are aggregated adherent cells known to mimic the complex intercellular interaction within the tumour microenvironment. The cytotoxicity of the bulb extract was determined using the MTT assay for monolayer culture and the Alamar Blue® assay for the spheroids of breast cancer cells, respectively. MTT assay results determined the IC50 values for monolayer culture at 25.17 µg/mL for MCF7 cells and 25.23 µg/mL for MDA-MB-231 cells. The Alamar Blue® IC50 results on MCF7 spheroids were recorded at 37.90 µg/mL and MDA-MB-231 spheroids at 48.72 µg/mL at 72 hours post-treatment. Thus, these findings further substantiate the cytotoxicity of E. americana ethanol extract towards the spheroids of MDA-MB-231. In conclusion, E. americana bulb extract has the potential to be developed as a phytotherapy for breast cancer.

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