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

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

Dried and deep frozen Glycyrrhiza root powder (60 mg) was homogenized with 5 ml 100% MeOH using a Turrax mixer (11,000 RPM) for five 20 s periods. Extracts were then vortexed vigorously and centrifuged at 3000g for 30 min to remove plant debris. For each root specimen, three biological replicates were provided and extracted in parallel under identical conditions (a total of 24 samples). For ROESY analysis, 4 ml was aliquoted and the solvent was evaporated under a stream of nitrogen till dryness. Dried extracts were resuspended with 800 ll 100% methanol-D4. After centrifugation (13,000g for 1 min), the supernatant was transferred to a 5 mm NMR tube. All spectra were recorded on a Varian VNMRS 600 NMR spectrometer operating at a proton NMR frequency of 599.83 MHz using a 5 mm inverse detection cryoprobe. 1 H NMR spectra were recorded with the following parameters: digital resolution 0.367 Hz/point (32 K complex data points); pulse width (pw) = 3 ls (45°); relaxation delay = 23.7 s; acquisition time = 2.7 s; number of transients = 160. 2D ROESY spectra was recorded using standard CHEMPACK 4.1 pulse sequences (gROESY) implemented in Varian VNMRJ 2.2C spectrometer software. ROESY experiment was conducted with a mixing time set for 300 ms. ROESY spectra were collected over a bandwidth of 6 ppm using 512 and 1024 complex data points in F1 and F2, respectively. Using a 1.31 s relaxation delay resulted in a total acquisition time of 28 min.