Purification and Characterization of the Principal Antimutagenic Bioactive as Ethoxy-Substituted Phylloquinone from Spinach (Spinacea oleracea L.) Based on Evaluation in Models Including Human Lymphoblast TK+/– Cells

During in vitro analysis, spinach ( Spinacea oleracea L.) leaf extracts displayed varying antimutagenicity when analyzed in models including human lymphoblast (TK +/– ) cell line (thymidine kinase gene mutation assay) and Escherichia coli MG1655 (rifampicin resistance assay) against chemically (ethyl methanesulfonate and 5-azacytidine) induced mutagenicity. Highest antimutagenicity was displayed by the quinone extract. The principal bioactive compound exhibited fluorescence in TLC at 366 nm (termed C4) resolved at R f 0.32 and t R 15.2 min in TLC and HPLC, respectively. On the TLC plate, three spots (C1–C3), observed at 254 nm, displayed comparatively lesser antimutagenicity. Furthermore, biochemical and spectroscopic analyses using MALDI-TOF MS and NMR indicated the nature of the potent compound (C4) as an ethoxy-substituted phylloquinone derivative [2-ethoxy-3-(( E )-3,7,11,15-tetramethylhexadec-2-enyl)naphthalene-1,4-dione]. The C4 compound did not display any cytotoxicity and hence possesses significant nutraceutical-based intervention possibility to combat the onset of mutation-associated disease(s).