Identification of prenylated pterocarpans and other isoflavonoids in Rhizopus spp. elicited soya bean seedlings by electrospray ionisation mass spectrometry

Abstract

Phytoalexins from soya are mainly characterised as prenylated pterocarpans, the glyceollins. Extracts of non-soaked and soaked soya beans, as well as that of soya seedlings, grown in the presence of Rhizopus microsporus var. oryzae, were screened for the presence of prenylated flavonoids with a liquid chromatography/mass spectrometry (LC/MS)-based screening method. The glyceollins I-III and glyceollidins I-II, belonging to the isoflavonoid subclass of the pterocarpans, were tentatively assigned. The formation of these prenylated pterocarpans was accompanied by that of other prenylated isoflavonoids of the subclasses of the isoflavones and the coumestans. It was estimated that approx. 40% of the total isoflavonoid content in Rhizopus-challenged soya bean seedlings were prenylated pterocarpans, whereas 7% comprised prenylated isoflavones and prenylated coumestans. The site of prenylation (A-ring or B-ring) of the prenylated isoflavones was tentatively annotated using positive-ion mode MS by comparing the (1,3) A(+) retro-Diels-Alder (RDA) fragments of prenylated and non-prenylated isoflavones. Furthermore, the fragmentation pathways of the five pterocarpans in negative-ion (NI) mode were proposed, which involved the cleavage of the C-ring and/or D-ring. The absence of the ring-closed prenyl (pyran or furan) gave exclusively -H(2) O(x,y) RDA fragments, whereas its presence gave predominantly the common RDA fragments.

Molecular Characterization of Soybean Pterocarpan 2-Dimethylallyltransferase in Glyceollin Biosynthesis: Local Gene and Whole-Genome Duplications of Prenyltransferase Genes Led to the Structural Diversity of Soybean Prenylated Isoflavonoids

“…1 ), which should be referred to as DMAPP:(−)-glycinol 2-dimethylallyltransferase (G2DT). Recent reports also illustrated that elicitor-treated soybeans accumulate glyceollins and several prenylated isoflavonoids, including 4-dimethylallylcoumestrol, 3′-dimethylallyldaidzein, 3′-dimethylallylgenistein and 8-dimethylallyldaidzein (Cheng et al 2011, Simons et al 2011) ( Fig. 1 ), further indicating the presence of several isoflavonoid PTs with varying substrate specificities and regiospecificities in soybean.…”

Identification of Glyceollin Metabolites Derived from Conjugation with Glutathione and Glucuronic Acid in Male ZDSD Rats by Online Liquid Chromatography–Electrospray Ionization Tandem Mass Spectrometry

“…17 For the GSH enzymatic products, the appearance of the m / z 149 fragment suggests that the oxygen and cysteinylglyceine dipeptide or cysteine are not located on the B fragments. More importantly, the A fragment expected at m / z 215 17,40 has been shifted to m / z 231 (Figure 2b), thus indicating that oxygen addition had occurred on the A fragment. On the basis of our evidence for initial epoxidation followed by GSH attachment, combined with the CID data presented above, we conclude that there are four possible attachment sites for the initial oxygenated glutathione conjugates of glyceollins (Scheme 1); the oxygenated cysteinylglyceine and oxygenated cysteinyl–glyceollin conjugates would maintain these same sites of conjugation.…”

Phytochemical analysis of isoflavonoids using liquid chromatography coupled with tandem mass spectrometry

“…A rare 2/4 rDA fragmentation, like that of isoflavan-derived flavonoids, has also been observed for pterocarpans in the negative mode (Simons et al 2011a).…”

“…Some of the various LC-MS/MS determination of isoflavonoids in the plant extracts were summarised along with their extraction methods in Table 3. Simons et al (2011a) identified isoflavonoids in Rhizopus spp. elicited soya bean seedlings by using RP-UPHLC associated with ESI-MS and successfully identified 13 isoflavones (Daidzin, Genistin, Glycitin, Malonyl-glycitin, Malonyl-daidzin, Acetyl-daidzin, 2 0 -Hydroxydaidzein, Malonylgenistin, Acetylgenistin, Glycitein, Daidzein, 2 0 -Hydroxygenistein and Genistein) and 5-pterocarpans (Glycinol, Glyceollidin I/II, Glyceollin III, Glyceollin II and Glyceollin I) in a single run (Simons et al 2011a).…”

“…Simons et al (2011a) identified isoflavonoids in Rhizopus spp. elicited soya bean seedlings by using RP-UPHLC associated with ESI-MS and successfully identified 13 isoflavones (Daidzin, Genistin, Glycitin, Malonyl-glycitin, Malonyl-daidzin, Acetyl-daidzin, 2 0 -Hydroxydaidzein, Malonylgenistin, Acetylgenistin, Glycitein, Daidzein, 2 0 -Hydroxygenistein and Genistein) and 5-pterocarpans (Glycinol, Glyceollidin I/II, Glyceollin III, Glyceollin II and Glyceollin I) in a single run (Simons et al 2011a). Lin et al (2000a, b) identified eight isoflavonoids calycosin-7-O-b-D-glucoside, calycosin-7-O-b-D-glucoside-6 00 -O-malonate, ononin, (6aR,11aR)-3-hydroxy-9,10-dimethoxypterocarpan-3-O-b-D-glucoside, calycosin, (3R)-7,2 0 -dihydroxy-3 0 ,4 0 -dimethoxyisoflavan-7-O-b-D-glucoside, formononetin-7-O-b-D-glucoside-60-O-malonate and formononetin.…”