The major metabolite detected was M5 with a molecular [MH]two ion at m/z 511, which was verified by precursor ion experiments scanning for m/z 269, the genistein anion. In addition, the corresponding sodium [M-2H+Na]2 and potassium [M-2H+K]2 
adduct ions at m/z 533 and m/z 549, respectively, ended up noticed (Figure 4A). The precise mass of the 117570-53-3 molecularion was determined to be 511.0652 by (ESI)-TOF experiments (Desk 1). The EPI scan at m/z 511 made, as illustrated in Determine 4B, fragment ions at m/z 269, 241, ninety seven, and 79. The correct mass fragment ions have been identified to be 269.0457, 241.0126, 96.9704, and seventy eight.9593, respectively (Desk 1). Based on those precise mass measurements we conclude that m/z ninety six.9704 and m/z 78.9593 represent the negatively charged phosphorous made up of fragment ions [PO3]2 and [H2PO4]two, therefore conclude that the major metabolite fashioned is a genistein7-O-(60-O-phospho)-b-D-glucoside. The proposed fragmentation pathway is depicted in Figure 5. Metabolite six and two happened in slight quantities and exhibited MS and MS/MS spectra analog to individuals of M5. Therapy of M6 with acid phosphatase yielded in genistein-forty nine-O-b-D-glucoside suggesting peak M6 to be genistein-49-O-(sixty-O-phospho)-b-Dglucoside. The third genistein-O-monophosho-hexoside M2 was so significantly not more characterised getting only present in trace quantities. With m/z 673 peak M4 showed the optimum molecular ion of all metabolites detected. This was verified by precursor ion experiments and detection of the corresponding sodium [M2H+Na]two and potassium [M-2H+K]2 adduct ions at m/z 695 and m/z 711, respectively (Determine 6A). Fragment ions at m/z seventy nine, m/z ninety seven and m/z 269 once yet again advise the existence of a phosphate group and the genistein aglycone. The prevalence of a fragment ion at m/z 269 signifies a genistein anion shaped by glycosidic bond cleavage and the decline of a phosphorylated disaccharide unit C12H21O13P (2404 Da), whilst the ion at m/z 403 outcomes from a neutral reduction of genistein (2270 Da) (Figure 6B). As a result we believe that the metabolite M4 is a genistein-O-phosphodissacharide. Desk 2 summarizes the experimental knowledge as nicely as the proposed composition of every metabolite. Electrospray mass spectra (QTrap method) of the deprotonated genistein metabolite M4 assigned as a genisteinphosphodisaccharide. (A) MS spectrum. (B) MS/MS spectrum of m/z 673. (C) Proposed constructions of the solution ions noticed in the MS/MS spectra. Hex, Hexose.
Our reports on the metabolic process of the isoflavone genistein in C. elegans exposed a novel biotransformation pathway. In addition, essential E. coli have been found as unable to make any of the genistein metabolites detected (data not demonstrated). All genistein metabolites shaped by C. elegans had been discovered as sugar conjugates, primarily genistein-O-glucosides. Similar glucosylation reactions of flavonoids in non-mammalian systems are primarily described for some microorganisms species, specially aerobic or facultative anaerobic rod-formed microorganisms these kinds of as Bacillus subtilis, Bacillus cereus, Xanthomonas campestris and Lactobacillus19756361 delbrueckii [17][18]. Far more lately Laing et al. [six] have been the initial to explain an analogous conjugation response in the nematode C. elegans exactly where a glucose moiety was released into the anthelmintic drug albendazole as a xenobiotic response and a selection of quercetin glycosides such as sulfate-glycosyl derivatives ended up recently located by Surco-Laos et al. [seven] as metabolites of the flavonoid quercetin by C. elegans. In our study, the dominant genistein metabolite in N2 C. elegans was reliably determined as a seven-O-phosphoglucoside.