psi-baptigenin and irilone

Starting with an isoflavone-rich red clover extract (RCE), this study expands on the DESIGNER approach to Deplete and Enrich Select Ingredients to Generate Normalized Extract Resources using countercurrent separation (CCS) methodology. A hydrostatic CCS (also known as centrifugal partition chromatography, CPC) technique was used to enrich and deplete selected bioactive isoflavones of RCE extracts. In order to efficiently prepare large enough DESIGNER extracts from RCE for biological testing including in vivo assays, it was necessary to choose a balance between resolution and a loading capacity of at least 1 g per separation for the selected solvent system (SS). Adding 3 mL of DMSO to the sample containing equal amounts of upper and lower phases of hexanes-ethyl acetate-methanol-water (HEMWat 5.5/4.5/5/5, v/v) allowed 1 g of RCE to be dissolved in the sample without disrupting the chromatographic resolution of the target isoflavones. CPC experiments using other solubility modifiers, acetone and acetonitrile indicated that these modifiers increase solubility significantly, even better than DMSO, but the separation of target compounds was sufficiently disturbed to be unacceptable for producing the desired DESIGNER extracts. The preparation of DESIGNER extracts was achieved with two sequential CPC separations. The first produced a biochanin A enriched fraction (93.60% w/w) with only small amounts of other isoflavones: 2.30% w/w prunetin, 1.17% w/w formononetin, and 0.12% w/w irilone. Gravimetric investigations of this step demonstrated the high efficiency of CCS technology for full and unbiased sample recovery, confirmed experimentally to be 99.80%. A formononetin enriched fraction from this first separation was re-chromatographed on a more polar HEMWat (4/6/4/6, v/v) SS to produce a formononetin enriched DESIGNER fraction of 94.70% w/w purity. The presence of the minor (iso)flavonoids: 3.16% w/w pseudobaptigenin, 0.39% w/w kaempferol, and 0.31% w/w genistein was also monitored in these fractions. Chromatographic fractions, combined fractions, and DESIGNER extracts were analyzed with quantitative

Topics: Countercurrent Distribution; Flavonoids; Genistein; Hexanes; Isoflavones; Methanol; Plant Extracts; Solvents; Trifolium

In addition to soya-derived preparations, red clover-based dietary supplements have gained considerable interest as an alternative isoflavone (IF) source. While metabolism and bioavailability of the main IF from both sources have already been investigated, studies are still lacking on the biokinetic behaviour of IF, which are present in red clover in minor amounts. In the present pilot study, in which seven volunteers ingested a single dose of a commercial red clover dietary supplement, we focused on the absorption of three such IF, irilone (IRI), prunetin (PRUN) and pseudobaptigenin (PBAP). The compounds were measured as aglycones after enzymatic hydrolysis. A single intake of an amount of as low as 3.8 mg IRI (out of 38.8 mg IF in total) resulted in an IRI plasma concentration of 0.35 (sd 0.16) mum at 6.5 h post-ingestion. Compared to the plasma concentrations found for daidzein (0.39 mum) and genistein (0.06 mum), expected to be the main IF metabolites in plasma, the present findings indicate that IRI might possess a relatively high bioavailability. Furthermore, PRUN and PBAP were detected in human plasma for the first time.

Topics: Adult; Biological Availability; Dietary Supplements; Female; Gas Chromatography-Mass Spectrometry; Humans; Isoflavones; Male; Pilot Projects; Tandem Mass Spectrometry; Trifolium