Crude methanol extract (ME) of broccoli (Brassica oleracea L.) sprout was fractioned by hexane, chloroform, ethyl acetate, butanol, and water. The contents of total polyphenols (19.89 mg GAE/g) and flavonoids (10.06 mg RE/g) were significantly higher in the butanol fraction (BF) than in the other fractions. The BF showed the highest DPPH (EC50 = 0.524 mg/mL) and ABTS (EC50 = 0.180 mg/mL) radical scavenging activities. High-performance liquid chromatography (HPLC) of crude ME showed that the most abundant phenolic compounds were rutin, quercetin, chlorogenic acid, catechin, and p-coumaric acid. The contents of quercetin, chlorogenic acid and p-coumaric acid were higher in the ethyl acetate fraction (EF) and BF than in the other fractions. Antioxidant activity and phenolic compound contents were correlated, suggesting that phenolics were responsible for the antioxidant activity. The hexane fraction (HF) and chloroform fraction (CF) decreased the viability of breast cancer stem cells (BCSCs), and the CF had the highest antiproliferative activity (IC50 = 69.47 mg/mL). The CF also suppressed the stemness characteristics of BCSCs and induced apoptotic cell death. The most abundant characteristic peak in CF was identified as oleic acid (area = 35.05%) by gas chromatography-mass spectrometry (GC–MS). Therefore, the broccoli sprout BF contained high levels of phenolic compounds that contributed to its antioxidant activity, and CF had a marked anti-proliferative effect on BCSCs.
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