Analysis of biliary metabolites of hypericin

To investigate in which form hypericin is present in the bile for interacting with the stones, CBD-ligated rats were prepared by ligation of the CBD in six normal rats with a non-absorbable silk suture. The resulting enlarged CBD were cannulated using polyethylene (PE) tubing (0.58 mm ID and 0.96 mm OD, Natume Co., Tokyo, Japan) for bile collection.

The CBD-ligated rats then received IV 1 × 10^-3 M hypericin at 10 mg/kg, and biliary juice was hourly collected from 0 to 9 h. Bile samples were transferred to a 96-well black polystyrene plate (Greiner Bio-One; Kremsmünster, Austria) and measured on a plate reader (FLUOstar OPTIMA, BMG Labtech, Offenburg, Germany) with excitation and emission filters of 485 and 590 nm wavelengths, respectively. The fluorescence concentrations versus time curves were built and the area under the curve from 0 up to 9 h (AUC0-9 h) was determined using the linear/logarithmic trapezoidal rule.

Bile samples were also analyzed by RP-HPLC using UV and fluorescence detection. Solutions of 1.3 × 10^-5 M hypericin either in DMSO/PEG-400/PG/H₂O (25%:25%:25%:25%, v/v/v/v) or bile were also analyzed.

The fluorescent HPLC peaks from bile analysis were collected and evaluated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as described previously[30]. After digestion and extraction of the tryptic peptides, the samples were dried down, desalted onto Millipore ZipTip C18 (Bedford, MA, United States) and analyzed by MS on a MALDI-TOF/TOF 4800 instrument (Applied Biosystems, Foster City, United States)[30]. Data interpretation was performed with the GPS Explorer software (Version 3.5), and database searching was done using the Mascot program (Version 2.2) (www.matrixscience.com).