Isolation and Characterization of Structurally Novel Antimutagenic Flavonoids from Spinach (Spinacia oleracea)

Abstract

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, (1)H and (13)C NMR, UV-vis spectroscopy, and mass spectrometry. All of these compounds were flavonoids and related compounds that could be attributed to five groups: (A, methylenedioxyflavonol 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%. IC(50) values (antimutagenic potencies) of the flavonol glucuronides ranged between 24.2 and 58.2 microM, 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 (IC(50) = 10.4 and 13.0 microM, respectively).

Extraction, separation, and detection methods for phenolic acids and flavonoids

“…It was demonstrated that UV spectra and first-order ESI mass spectra allowed a fast characterisation of flavonoids even if reference compounds were not at hand or available. Identification of phenolics separated after HPLC was also carried out using fast atom bombardment mass spectrometry [154,155] and electron impact mass spectrometry [154]. On the other hand, matrix-assisted laser desorption/ionisation mass spectrometry has been employed for qualitative and quantitative analysis of anthocyanins in foods [156].…”

Determination of the glycosylation site of flavonoid monoglucosides by metal complexation and tandem mass spectrometry

“…Despite these inroads, mass spectrometric analysis has not yet reached the point where de novo identification of flavonoids is possible. Analysis of flavonoids in plant extracts [34 -40], foodstuffs [41][42][43][44][45][46], and human biological fluids [47][48][49] have been reported, but the identity of flavonoids in these complex matrices generally must be confirmed by comparison with commercial standards. Otherwise only tentative identifications can be made, particularly in terms of saccharide location and identity, unless supplementary analytical methods, such as UV, FTIR, or NMR spectroscopy, are employed.…”

Flavonoid glucuronides isolated from spinach inhibit IgE-mediated degranulation in basophilic leukemia RBL-2H3 cells and passive cutaneous anaphylaxis reaction in mice

“…There are only a few papers describing the biological effects of flavonoids isolated from spinach, such as antioxidative and antimutagenic activities [5][6][7]. Very recently, Ishida et al [19] reported the inhibitory activity of aqueous spinach extract on the degranulation of RBL-2H3 cells.…”

“…In the previous papers relating to spinach constituents, it contains a various compound such as nutrients, vitamins, minerals and polyphenolic compounds. To date, it has been reported that spinach has antioxidant effect, anti-mutagenic and anti-inflammatory effects [5][6][7][8][9][10][11]. The antioxidant effect of spinach is reported to be mainly due to the flavonoid compounds such as spinacetin glucoside, patuletin glucoside and dioxol flavonoid [5,7,10].…”