Unequivocal glycyrrhizin isomer determination and comparative in vitro bioactivities of root extracts in four Glycyrrhiza species

Farag, Mohamed A.; Porzel, Andrea; Wessjohann, Ludger A.

Human prostate PC3 cancer cell line was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, (DSMZ ACC# 465) and the HT29 colon cancer cell line was obtained from the medical immunology department at Martin Luther-Universita¨t Halle-Wittenberg (Prof. Seliger). The cells were grown as monolayers in adherent cell lines and were routinely cultured in RPMI (Roswell Park Memorial Institute) 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% L-glutamine in 75 cm 2 polystyrene flasks (Corning Life Sciences, UK) and maintained at 37°C in a humidified atmosphere with 5% CO 2 . Cells were plated at a density of 1 · 10 4 /well in 96-well plates. They were allowed to attach to the plate for 24 h. After 24 h, the media were replaced with RPMI media containing resin extracts. Three concentrations were used (5, 10, and 20 lg/ml) from each extract. Dried extracts prepared as in section 3.3 were initially dissolved in DMSO at a concentration of 2 mg/ml and further diluted with RPMI medium. The DMSO concentration in the assay did not exceed 0.1% and was not cytotoxic to the tumor cells. After 72 h, the medium was taken out, 100 ll of XTT-solution (2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) (Roche Applied Science, Mannheim, Germany) was added to each well, and plates were incubated at 37°C for another 4 h at a (final concentration 0.3 mg/ml) . Absorbance was measured at 490 nm against a reference wavelength at 650 nm using a microplate reader (Beckman Coulter, DTX 880 Multimode Reader). The mean of triplicate experiments for each dose was used to calculate the IC 50 and repeated in 2 passages for each cancer cell line. Digitonin was used as a positive drug control.