Immunoanalysis of isoflavonoids in Pisum sativum and Vigna radiata

Radioimmunoassays (RIAs) combined with liquid chromatography were used for study of isoflavonoids in seeds of pea Pisum sativum and mung bean Vigna ra diata. Radioimmunoassays with these specifities were used: (I) daidzein and its 4'-derivatives (e.g. formononetin); (2) daidzein and its 7-derivatives (e.g. daidzin, isoformononetin); (3) genistein and its 4'-derivatives (e.g . biochanin A); and (4) genistein and its 7-derivatives (e.g. genistin, pru netin). Dormant or germinating seeds were extracted with 80% ethanol. Immun oreactivities were measured either in crude extracts or after chromatograph ic fractionation by HPLC (reversed phase, octadecylsilica). Chromatographic mobilities of immunoreactive fractions were compared to those of daidzein, daidzin, formononetin, isoformononetin, genistein, genistin biochanin A an d prunetin standards. Extracts from Vigna radinfa contained daidzein, genis tein and their 7-0 glucosides, daidzin and genistin, respectively. No immun oreactivity was recorded in HPLC fractions corresponding to glycosides in e xtracts from P. sativum, but the methods sensitive to 7-derivatives of daid zein and genistein showed peaks with chromatographic mobilities identical t o those of the 7-methoxyderivatives, isoformononetin and prunetin, respecti vely. In additional experiments, the pea extracts were fractionated either by thin layer chromatography (TLC) on silica or by ion-exchange TLC on amin osilica. Identity of the daidzein-7 and genistein-7 immunoreactive entities with isoformononetin and prunetin, respectively, was confirmed by the iden tical chromatographic behavior in all these different chromatographic syste ms. (C) 1999 Elsevier Science Ireland Ltd. AII rights reserved.