Preparative enzymatic synthesis and HPLC analysis of rhapontigenin: applications to metabolism, pharmacokinetics and anti-cancer studies.

OBJECTIVE

A facile method was established to enzymatically synthesize rhapontigenin from the glycosylated parent compound rhaponticin. A novel and simple high-performance liquid chromatographic method was developed for the determination of rhapontigenin. The assay was successfully applied to both the in vitro and in vivo metabolic kinetic study of rhapontigenin.

METHODS

Serum, or microsomes (0.1 mL) was precipitated with acetonitrile after addition of the internal standard, daidzein. Separation was achieved on an amylose tris 3,5 dimethylphenylcarbamate column (150 x 4.6 mm, ID, 5m) with UV detection at 324 nm. Hep G2 hepatoma cells were treated with rhapontigenin or rhaponticin (0-250 microg/mL) and cell viability was measured.

RESULTS

The calibration curves were linear ranging from 0.5 to 100 micromg/mL. The mean extraction efficiency was > 99%. Precision of the assay (coefficient of variation) was < 5%, including the limit of quantitation (0.5 microg/mL). Bias of the assay was lower than 5%. The limit of detection was 100 ng/mL for a 0.1 mL sample. One glucuronidated metabolite of rhapontigenin has been identified. Preliminary pharmacokinetic data revealed the presence of a glucuronidated metabolite in the serum and a terminal elimination t1/2 of approximately 6 h. Rhapontigenin demonstrated concentration-dependent anti-cancer activity with an IC50 115 microg/mL in HEP G2 cells while rhaponticin showed no activity across the concentrations tested in vitro.

CONCLUSIONS

The preparative enzymatic synthesis method has demonstrated utility to provide sufficient rhapontigenin for pharmaceutical studies. Rhapontigenin is an active anti-cancer compound. The developed HPLC assay is sensitive, reproducible and accurate and can be applied to pharmacokinetic and metabolism studies.