High performance liquid chromatogtaphy determination of cucurbitacins in the roots of Wilbrandia ebracteata Cogn.

Roots of Wilbrandia ebracteata Cogn., Cucurbitaceae, used in folk medicine,for treatment of rheumatic disease, are rich in cucurbitacins. Dihydrocucurbitacin B is, the most abundant cucurbitacin while cucurbitacin B is a minor component. A reverse-phase HPLC system was developed for simultaneous quantitative assay of these cucurbitacins in the roots. The optimised,experimental conditions were acetonitrile/H(2)0 40:60, flow-rate 1.2 mL/min'., detection at 230 nm and isocratic elution. A variety of sample preparation modes were,tested and the extraction, with dichloromethane under reflux gave better results. The validation process included linearity, accuracy, repeatability and intermediate precision. The calibration curve of dihydrococurbitacin B was linear from 40.00, to 400 mu g/mL, the recovery was 95.5 +/- 3.01%, the intermediate precision was found to be 1.64% and the repeatability varied between 1.30 to 2.05%. The calibration curve of cucurbitacin B was linear from 4.00 to 240 mu g/mL, intermediate precision was found to be 2.29% and repeatability varied between 1.03 to 2.95%. Analysis of the same specimen of W ebracteata every year from 2002 to 2005 revealed a great rise on the cucurbitacin B concentration after-the root was attacked by an herbivore.