An enzyme-linked immunosorbent assay for the measurement of plasma flavonoids in mice fed apigenin-C-glycoside.

ABSTRACT

BACKGROUND: In the Chenopodiaceae family, the apigenin flavonoids vitexin-2-O-xyloside (VOX) and vitexin-2-O-rhamnoside (VOR) are important chemopreventive components. To investigate their bioavailability in in vivo animal studies an enzyme-linked immunosorbent assay (ELISA) method has been developed. RESULTS: The ELISA was based on polyclonal antibodies elicited in mice by injecting, as an immunogen, 4',6″-O-biapigenin (hinokiflavone, HF) conjugated to bovine serum albumin (BSA-HF). A second immunogen was synthesised by coupling an equimolar mixture of VOX and VOR to BSA (BSA-F1). The BSA-HF elicited a significant antibody response, due to 17 HF hapten groups, coupled to each BSA molecule, whereas BSA-F1 provided a very low antigenicity in respect to control animals. Antiserum raised against BSA-HF showed an antibody titre of 11600. Antibodies were found to be specific for the flavonols. Our results show that VOX and its metabolic products reached the concentration of 3.42 ± 0.72 µg mL⁻¹ in plasma of VOX fed animals, at the net of the control value. CONCLUSIONS: By using the ELISA, the concentration of apigenin flavonoids and their metabolites can be detected in VOX- or VOR-supplemented animals. The assay represents a useful tool for rapid screening to compare bioavailability of apigenin flavonoids in respect to control animals.