Antioxidant Activity and Inhibitory Effects of Black Rice Leaf on the Proliferation of Human Carcinoma Cells

The leaves of black rice, well-known as postharvest agricultural waste, contain a rich source of antioxidants with multiple benefits for human health. In the present study, the ethyl acetate fraction obtained from black rice leaf was separated into five subfractions using Sephadex LH-20 column chromatography, and their antioxidant and anticancer activities were investigated. The results revealed that among all the subfractions, subfraction 5 (Sub5) showed the highest total phenolic and flavonoid values. The antioxidant activity was also superior in Sub5 (the IC₅₀ values are 3.23, 31.95, and 72.74 μg/mL, in the DPPH, ABTS, and reducing power assays, respectively) compared to the other subfractions. All subfractions, in a time-dependent manner, inhibited the proliferation of hepatoma (HepG2), breast (MCF-7), and colorectal (Caco-2) cancer cells, especially the Sub5. Thus, Sub5 was employed to conduct the cell cycle and cell apoptosis by flow cytometry. Sub5 significantly increased the accumulation of cells at the Sub-G1 phase in HepG2 cells (44.5%, at 48 h). Furthermore, it could trigger annexin V-detected apoptosis through mitochondrial and death receptor pathways accompanied by the suppression of PI3K/Akt and Erk signaling pathways. In addition, HPLC-DAD-MS/MS was conducted to characterize the bioactive constituents in the most potent antioxidant, cytotoxic, and apoptosis-inducing subfraction. Conclusively, Sub5 may have high potential as functional dietary supplements to inhibit the development of HepG2 liver cancer.