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In the search for new anticancer agents of natural origin against breast and lung cancer (ZR-75 and A549 cancer cell lines, respectively), plant-associated endophytes could be a good source for bioactive secondary metabolites. Twenty six endophytes were obtained from four different Jordanian plants; Anchusa strigosa, Anthemis palestina, Euphorbia peplus and Rumex cyprius. Internal transcribed spacer (ITS) gene sequencing was implemented to identify the obtained endophytes. Based on their biological activity and chemical profile, three endophytes namely Curvularia australiensis, Chaetomium subaffine and Fusarium acuminatum were chosen for the scale-up. These endophytes were cultured in liquid and rice media at different time periods to optimise their growth and production of compounds, employing both NMR and mass spectrometry-based metabolomics. The medium that afforded better yield, more chemical diverse extract and more potent biological activity was chosen for scaling-up purposes. Each of the scaled-up extracts was subjected to liquid-liquid partitioning followed by fractionation using a high-throughput flash-chromatography system. The fractions obtained from the first chromatography step were tested in-vitro against both breast and lung cancer (ZR-75 and A549 cell lines, respectively) and analysed using both proton nuclear magnetic resonance (NMR) and liquid chromatography-high resolution mass spectrometry (LC-HRMS). The HRMS data were processed with MZmine then subjected to Orthogonal Partial Least Square-Discriminant Analysis (OPLS-DA). The OPLS-DA results pinpointed the biologically active secondary metabolites. Metabolomics-guided isolation work targeted the bioactive secondary metabolites. As a result, five new compounds and ten known compounds were obtained from the three scaled-up endophytes. The isolated compounds were elucidated by employing 1D and 2D NMR then tested against ZR-75 and A549 cell lines. Twelve compounds were found active against ZR-75 cell line, which included five new compounds. Six compounds were found active against A549 cell line that included one of the new natural products isolated. |
