**5. Isolation and structural determination of phenolic compounds from** *G. uralensis*

The roots of *G. uralensis* (1.2 kg) were extracted with EtOAc (5.5 L) at room temperature for 7 days. The extract was then filtered and concentrated (74.0 g). The EtOAc extract of *G. uralensis* roots exhibited potent PPAR-γ ligand-binding activity and its relative luminescence intensity was 2.8 at a sample concentration of 30 μg/mL. This was almost equivalent to that of 1.0 μM TRG. The EtOAc extract was chromatographed on a silica gel column eluted with CHCl<sup>3</sup> –MeOH gradients (19:1; 9:1; 4:1) and finally with MeOH to provide four fractions. PPAR-γ ligand-binding activity was concentrated in the CHCl<sup>3</sup> -MeOH (19:1) eluate (relative luminescence intensity of 2.8 at 30 μg/mL). The CHCl<sup>3</sup> -MeOH (19:1) eluate (55.4 g) was subjected to multiple chromatographic steps on a silica gel column eluted with CHCl<sup>3</sup> -MeOH, an ODS silica gel column eluted with MeOH-H2 O and MeCN-H2 O, and to reversed phase HPLC using MeOH-H2 O and MeCN-H2 O mobile phases to obtain compounds **40**–**52** (22.2, 28.3, 58.7, 225, 80.7, 12.1, 43.5, 17.5, 11.8, 74.5, 51.0, 22.1, and 40.1 mg, respectively). Compounds **41**–**52** were identified by comparison of their physical and spectral data with those of reported compounds as dehydroglyasperin C (**41**) [28], glyasperin D (**42**) [29], glycycoumarin (**43**) [30], glycyrin (**44**) [31], glyasperin B (**45**) [29], glycyrol (**46**) [32], isoglycyrol (**47**) [32], glicoricone (**48**) [33], licoricone (**49**) [34], licocoumarone (**50**) [30], gancaonin I (**51**) [35], and liquiritigenin (**52**) [36] (**Figure 8**). This was the first isolation of glyasperin B (**45**) from *G. uralensis*. The structure of the new compound, **41**, was determined by 1D and 2D NMR spectroscopic analysis and

**Figure 8.** Structures of **40–52** isolated from *G. uralensis* roots [2]. Values in parentheses are the relative luminescence intensities at 5 μg/mL.

that the two non-cyclic prenyl moieties were necessary for the potent activity of these compounds (**Figure 7**). Taking together all the above data, the PPAR-γ ligand-binding activity of the phenolic compounds was affected by slight differences in the substitution groups on the aromatic rings.

\*\* (*p* < 0.01) as determined by Dunnett's multiple comparison test.

84 Biological Activities and Action Mechanisms of Licorice Ingredients

**Figure 6.** PPAR-γ ligand-binding activity of compounds **1–39** at 2 ( ) and 10 ( ) μg/mL with a GAL-4-PPAR-γ chimera assay [1]. Troglitazone (TRG) at 0.5, 1.0, and 2.0 μM was used as a positive control, and dimethyl sulfoxide at 1 mL/L was used as a solvent control. Values are means ± SD, *n* = 4 experiments. Statistical significance is indicated by \* (*p* < 0.05) or HRESIMS as 3-(2,4-dihydroxyphenyl)-5,7-dimethoxy-6-(3-methyl-2-butenyl)-2H-chromene and was named dehydroglyasperin D.
