Apoptotic Effects of Agapanthus africanus Extracts and Identification of Volatile Compounds from the n-Butanol Fraction

Abstract

<i>Agapanthus africanus</i> (L.) Hoffmanns. is a medicinal plant traditionally used in South Africa for its promise as a source of bioactive compounds with anticancer properties. This study aimed to investigate the apoptotic effects of <i>A. africanus</i> fractions on cancer cell lines and to identify the bioactive phytochemical constituents using gas chromatography-mass spectrometry analysis. To test for cytotoxicity, MCF-7, A549, and HeLa cancer cells were treated with crude extract, <i>n</i>-hexane, <i>n</i>-butanol, dichloromethane, and aqueous fractions of <i>A. africanus</i> extracts at different concentrations (0.00-1000 µg/mL). Total apoptosis was quantified using Annexin V/PI staining. The 4',6-diamidino-2-phenylindole was used to detect nuclear morphological changes and the Caspase-GLO 3/7 assay was employed to check the caspase activation in the cancer cells. Expression of apoptosis-related (<i>caspase-3</i>, <i>bax</i>, <i>bcl-2</i>) genes was evaluated using real time-polymerase chain reaction. The crude extract of <i>A. africanus</i> exhibited dose-dependent cytotoxicity against MCF-7, A549, and HeLa cells, with IC₅₀ values of 130 µg/mL, 380 µg/mL, and <125 µg/mL, respectively. Among the tested fractions, the <i>n</i>-butanol fraction showed cytotoxicity towards MCF-7 cells with an IC₅₀ value of <870 µg/mL. In contrast, <i>n</i>-hexane, dichloromethane and the aqueous fractions exhibited higher IC₅₀ values against cancer cells. Flow cytometry analysis, which was applied to quantify total apoptosis, revealed that the crude extract of <i>A. africanus</i> induced apoptosis by (~60%) compared to the <i>n</i>-butanol fraction, which exhibited a moderate apoptotic effect (~27%). DAPI nuclear staining showed nuclear shrinkage and chromatin condensation in the MCF-7 cell line, whereas in Caspase-GLO 3/7, the crude extract and <i>n</i>-butanol fraction resulted in significant luminescence, indicating activation of caspase-3/7. Caspase-3/7 analysis showed <i>A. africanus</i> treatments produced varying levels of apoptotic activation. The crude extract increased caspase activity by 2.9-fold, while the <i>n</i>-butanol fraction induced a 1.7-fold rise compared with untreated cells. GC-MS chromatograms detected and identified 16 compounds in the fractionated <i>n</i>-butanol and 23 compounds from the crude extract of <i>A. africanus</i>. The major compounds identified from the <i>n</i>-butanol fraction included <i>n</i>-hexadecanoic acid; α-tocopherol and 9,12,15-octadecatrienoic acid, while the GC-MS profile of the crude extract was dominated by 6,10,14-trimethylpentadecan-2-one; 1,3,5-Triphenylcyclohexane and phytol. The study indicates the pro-apoptotic potential of <i>A. africanus</i>, particularly in its crude form, supporting its ethnopharmacological use and suggesting its relevance as a candidate for anticancer drug discovery.