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Hibiscus sabdariffa calyx is a rich source of anthocyanins and other bioactive compounds but no study reported the effects of experimental conditions on the extraction of these chemical compounds. Therefore, the effects of time and extraction temperature on the bioactive compounds and antioxidant activity of Hibiscus sabdariffa calyx were evaluated. In addition, the effects of copigmentation and pH on the stability of anthocyanins were assessed and the cytotoxic effects (LC 50 , IC 50 , and GC 50 ) of the extracts were determined in relation to tumor cell lines - Caco-2, HepG-2, HCT8, and A549. The temperature significantly influenced the total anthocyanins and flavonoids contents. The interaction between time/temperature influenced the total phenolic content and ascorbic acid. The t 1/2 and the percentage of colour retention decreased markedly at temperatures above 80 °C. Variations in pH conserved the antioxidant activity of the anthocyanins, and the protonation-deprotonation process of the extract was reversible. The treatment of cells with purified anthocyanin extract or crude extracts at 5-800 μg mL -1 did not show significant cytotoxic effects on the cell lines, corroborating the chemical antioxidant effect of the extracts (DPPH assay). Cyanidin-3-glucoside, delphinidin-3-sambubioside, delphinidin-3-glucoside, and cyanidin-3-sambubioside were identified in the extracts by LC-ESI-MS., (Copyright © 2018 Elsevier Ltd. All rights reserved.)