Isolation and characterization of structurally novel antimutagenic flavonoids from spinach (Spinacia oleracea)

Thirteen compounds, isolated from spinach (Spinacia oleracea), acted as antimutagens against the dietary carcinogen 2-amino-3-methylimidazo [4,5-f]quinoline in Salmonella typhimurium TA 98. The antimutagens were purified by preparative and micropreparative HPLC from a methanol/water (70:30, v/v) extract of dry spinach (commercial product) after removal of lipophilic compounds such as chlorophylls and carotenoids by solid-phase extraction (SPE). Pure active compounds were identified by instrumental analysis including FT-IR, H-1 and C-13 NMR, UV-vis spectroscopy, and mass spectrometry. All of these compounds were flavonoids and related compounds that could be attributed to five groups: (A, methylenedioxyflavonal glucuronides) 5,3 ' -dihydroxy-4 ' -methoxy-6,7-methylenedioxyflavonol 3-O-beta -glucuronide (compound 1), 5,2 ' ,3 ' -trihydroxy-4 ' -methoxy-6,7-methylenedioxyflavonol 3-O-beta -glucuronide (compound 2), 5-hydroxy-3 ' ,4 ' -dimethoxy-6,7-methylenedioxyflavonol 3-O-beta -glucuronide (compound 3); (B, flavonol glucuronides) 5,6,3 ' -trihydroxy-7,4 ' -dimethoxyflavonol 3-O-beta -glucuronide (compound 4), 5,6-dihydroxy-7,3 ' ,4 ' -trimethoxyflavonol 3-O-beta -glucuronide (compound 5); (C, flavonol disaccharides) 5,6,4 ' -trihydroxy-7,3 ' -dimethoxyflavonol 3-O-disaccharide (compound 6), 5,6,3 ' ,4 ' -tetrahydroxy-7-methoxyflavonol 3-O-disaccharide (compounds 7 and 8); (D, flavanones) 5,8,4 ' -trihydroxyflavanone (compound 9), 7,8,4 ' -trihydroxyflavanone (compound 10); (E, flavonoid-related compounds) compounds 11, 12, and 13 with incompletely elucidated structures. The yield of compound 1 was 0.3%, related to dry weight, whereas the yields of compounds 2-13 ranged between 0.017 and 0.069%. IC50 values (antimutagenic potencies) of the flavonol glucuronides ranged between 24.2 and 58.2 µM, whereas the flavonol disaccharides (compounds 7 and 8), the flavanones (compounds 9 and 10), and the flavonoid-related glycosidic compounds 11-13 were only weakly active. The aglycons of compounds 7 and 8, however, were potent antimutagens (IC50 = 10 4 and 13.0 µM, respectively).