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

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

We have recently reported on the use of 1 H NMR for the metabolic fingerprinting of G. glabra, G. uralensis and G. inflata extracts, targeting its secondary metabolites [17] . G (Fig. 1 ) was identified in these extracts from its well resolved singlet signals at d 0.82 (Me-28), 0.85 (Me-24), 1.08 (Me-23), 1.13 (Me-25& 26), 1.16 (Me-29) and 1.41 (Me-27), (Fig. 2C) . In the sugar region, signals at d 4.49 d (7.3 Hz) and 4.67 d (7.7 Hz) were assigned to anomeric protons of glucuronic acid moieties at H-1 0 and H-1 00 position in glycyrrhizin, respectively [17] . For detailed description on extracts chemical composition and glycyrrhizin concentration, see Farag et al. [17] . NMR signals were assigned using a combination of 2D NMR experiments ( 1 H, 1 H COSY, HSQC, and HMBC). Nevertheless, utilizing 1 D NMR and these 2D-experiments, G isomer conformational assignment could not be successfully confirmed in most licorice extracts, due to crowded 1 H NMR spectra with matrix signals which do not allow detection of signals of 18aor 18b-G. 2D ( 1 H-1 H) ROESY, a method based on proton-proton dipolar relaxation through space, was considered suitable to distinguish between 18-a and b isomers. ROESY spectra for a-and b-glycyrrhetic acid standard show distinctive crosspeaks between H-18 and its neighboring protons indicative for each isomer configuration. While the a-isomer exhibits 1,3-diaxial interaction between H-18 (d 2.37) and CH 3 -27 (d 1.39) as well as CH 3 -29 (d 1.24), the b-isomer shows interaction between H-18 (d 2.18) and equatorial CH 3 -28 appearing at d 0.82 ( Fig. 2A and B ). ROESY spectra acquired from all licorice samples revealed that the presence of G in its b-form coincides with standard GA b-isomer crosspeak patterns (Fig. 2C) . The variance was assessed by analyzing samples from three biological replicas for each specimen. Analytical reproducibility was further assessed by measuring triplicate NMR measurements over 2 days for the biological sample GG1 and showing almost superimposed crosspeak results (data not shown). These results confirm previous reports highlighting that the b-isomer is the natural form of glycyrrhizin in G. glabra and extends to allied species studied in this paper such as G. inflata and G. uralensis.