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

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

All reagents and solutions were prepared according to the protocols established by Cayman Chemicals (Ann Arbor, MI, USA) for the COX-1 inhibition assay. MeOH extract prepared as above was dissolved in neat DMSO and diluted in reaction buffer to a final DMSO concentration of 1% (v/v). Extracts were tested at a dose of 100, 150 and 200 lg/ml; 3 replicates for each concentration. Reactions were conducted with COX-1 enzyme in the presence of heme as a co-factor. The enzymes were incubated at 37°C for 15 min with serial dilutions of the liquorice extract or reaction buffer to determine 100% enzyme activity (positive control), and heat inactivated enzymes were used to as negative controls (0% enzyme activity). Arachidonic acid (100 lM) was added to each well and the fluorescence product of the reaction was measured at 670 nm using microplate reader. Percent inhibition of the COX enzymes was determined by comparing the extract loaded wells with the positive and negative controls. Commercially available COX-1 inhibitor (SC-560) (Cayman Chemicals, Ann Arbor, MI, USA) with an IC 50 value of 100 lM was used as positive reference for comparison.