Phytochemical analysis and anticancer activity of fruit extracts of Southern African pomegranate (Punica granatum) 'Wonderful' cultivar

Due to its superior nutritional, health promoting and disease preventing qualities, the Punica granatum is referred to as a "Super Fruit” and has been traditionally used for its therapeutic properties including cancer prevention. Although this plant is grown globally, phytochemical profile and their biological properties varies based on the pedoclimatic variables, fruit's phenogenotype, stage of ripeness, pre- and post-harvest circumstances, and extractive solvent, even among the same cultivars grown under various conditions. Here, we investigated the phytochemical composition and anticancer potency of three different aril solvent extracts (methanol, chloroform, and ethyl acetate) of South African ‘Wonderful’ cultivar against human breast cancer MCF-7 cell line. The identification of major phytochemical constituents of three different extracts was performed by UHPLC-Q-TOF-MS². Total phenolic and flavonoid content were determined using the Folin–Ciocalteau and aluminium chloride colorimetric methods, respectively. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction and adenosine triphosphate (ATP) content assays. The apoptotic effects were determined by morphological analysis (phase contrast and LIVE DEAD assay) and FITC-Annexin/ Propidium Iodide flow cytometry assay, Cytochrome-c release, caspase activation assay and ratio of Bax/Bcl-2 expression. UHPLC-Q-TOF-MS² analysis of extracts revealed the presence phenolic acids, flavonoids, anthocyanins, tannins, coumarins, gallotannins, lignan, and phthalide. The total phenolic and flavonoid content was determined to be highest for methanol extract (91.78±1.55 mg GAE/g DM and 43.03±2.63 mg QE/g DM respectively). This correlated with its highest cytotoxic activity against MCF-7 cells compared to other two solvent extracts with IC₅₀ concentration ∼289 and ∼229 μg/mL at 24 and 48 h respectively. After 48 h, the apoptotic cell population were significantly increased with 400 μg/mL methanol extract treatment as observed via morphological changes and FITC-Annexin/ Propidium Iodide assay. The methanol extract significantly induced apoptosis via the release of cytochrome-c, increase in ratio of Bax to Bcl-2 and caspase-8 and 9 activations. Based on the present findings, we conclude that South African P. granatum ‘Wonderful’ cultivar had the potential as cytotoxic and apoptosis inducing anticancer agent, which is worth investigating in detail.