**3. Isolation and structural determination of phenolic compounds from** *G. glabra*

insulin resistance syndrome. We found that the EtOH extract of *Glycyrrhiza glabra* roots and the EtOAc extract of *G. uralensis* roots exhibited higher activity than did the other materials tested. Bioassay-guided fractionation of these extracts resulted in the isolation of 52 phenolics,

In this chapter, we describe the results of the bioassay-guided fractionation of *G. glabra* and

PPAR-γ ligand-binding activity was assessed using a GAL-4-PPAR-γ chimera assay system (**Figure 1**) [3]. CV-1 monkey kidney cells from the American Type Culture Collection (ATCC) were suspended in Dulbecco's Modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 50 IU/mL Penicillin G sodium salt, 50 μg/mL streptomycin sulfate, and 37 mg/L

tial medium (MEM) and pM-hPPAR-γ and p4 × UASg-tk-luc were transfected into cells using LipofectAMINE PLUS (Gibco). pM and p4 × UASg-tk-luc were transfected into CV-1 cells as a mock control. Twenty-four hours after transfection, the medium was changed to DMEM containing 10% charcoal-treated FBS [4] and the cells were further cultured for 24 h. The cells were then washed with phosphate-buffered saline containing Ca2+ and Mg2+, and luciferase activity was measured using LucLite (Perkin-Elmer). Luminescence intensity was measured using a TopCount Microplate scintillation/luminescence counter. PPAR-γ ligand-binding activity was expressed as the relative luminescence intensity (test group/control group) deter-

/air at 37°C for 24 h. Cells were washed with OPTI-minimum essen-

cells/well

ascorbic acid. The cells were then inoculated into a 96-well culture plate at 6 × 10<sup>3</sup>

*G. uralensis* roots using a GAL-4-PPAR-γ chimera assay method.

including 11 novel ones [1, 2].

and incubated in 5% CO2

mined for each sample.

**Figure 1.** GAL4-PPAR-γ chimera assay system.

**2. PPAR-γ ligand-binding activity**

78 Biological Activities and Action Mechanisms of Licorice Ingredients

The roots of *G. glabra* (4.0 kg) were extracted twice with 20 L of 95% EtOH for 2 h at 45°C. The extracts were combined and concentrated under reduced pressure to give a 95% EtOH extract (120 g). The extract exhibited a relative luminescence intensity of 2.2 at a sample concentration of 5 μg/mL, indicating a PPAR-γ ligand-binding activity that was almost as strong as that of 0.5 μM troglitazone (TRG), a potent synthetic PPAR-γ agonist. The extract was chromatographed on a silica gel column eluted with CHCl<sup>3</sup> -MeOH gradients (19:1; 9:1; 2:1) and finally with MeOH. After removal of solvent, the fraction eluted with CHCl<sup>3</sup> -MeOH (19:1) yielded 85 g of material that showed notable PPAR-γ ligand-binding activity (**Figure 2**). A series of chromatographic separations were then performed, resulting in 10 new phenolic compounds [**1** (8.0 mg), **2** (13.8 mg), **3** (1.6 mg), **4** (6.9 mg), **5** (18.5 mg), **6** (8.6 mg), **7** (18.5 mg), **8** (4.9 mg), **9** (7.3 mg), **10** (30.2 mg)] (**Figure 3**). In addition, 29 known phenolic compounds were obtained and identified by comparison of physical and spectral data with those reported in the literature. The following known phenolic compounds were identified in the CHCl<sup>3</sup> -MeOH (19:1) fraction: echinatin (**11**, 5.4 mg) [5], lichocalcone B (**12**, 17.3 mg) [6], morachalcone A (**13**, 14.1 mg) [7], 2′,3,4′-trihydroxy-3′-γ,γ-dimethylallyl-6′′,6′′-dimethylpyrano[2′′,3′′:4,5]chalcone (**14**, 17.4 mg) [8], 1-(2′,4′-dihydroxyphenyl)-2-hydroxy-3-(4′′-hydroxyphenyl)-1-propanone (**15**, 5.8 mg) [9], kanzonol Y (**16**, 2.5 mg) [10], (3*R*)-vestitol (**17**, 10.5 mg) [11], (3*R*)-2′,3′,7-trihydroxy-4′-methoxyisoflavan (**18**, 8.1 mg) [12], kanzonol X (**19**, 37.1 mg) [10], glabridin (**20**, 193 mg) [13], 4′-*O*-methylglabridin (**21**, 11.4 mg) [14], 3′-hydroxy-4′-*O*-methylglabridin (**22**, 54.7 mg) [15],

**Figure 2.** PPAR-γ ligand-binding activity of *G. glabra* extract and fractions [1]. PPAR-γ ligand-binding activity of the extract and fractions (5 μg/mL), as well as that of troglitazone (0.5 μM) used as a positive control, was measured using a GAL-4-PPAR-γ chimera assay. Statistical significance is indicated as \* (*p* < 0.05) or \*\* (*p* < 0.01) as determined by Dunnett's multiple comparison test.

**Figure 3.** Structures of **1–10** isolated from *G. glabra* roots [1].

hispaglabridin A (**23**, 13.7 mg) [14], hispaglabridin B (**24**, 10.7 mg) [14], glabrene (**25**, 28.8 mg) [16], kanzonol W (**26**, 3.0 mg) [10], glabrocoumarin (**27**, 16.4 mg) [17], shinpterocarpin (**28**, 41.0 mg) [18], *O*-methylshinpterocarpin (**29**, 32.4 mg) [18], licoagrocarpin (**30**, 6.1 mg) [19], licoflavanone A (**31**, 4.8 mg) [20], glabrol (**32**, 13.5 mg) [13], shinflavanone (**33**, 7.8 mg) [18], euchrenone a5 (**34**, 1.5 mg) [21], xambioona (**35**, 8.0 mg) [22], gancaonin L (**36**, 8.8 mg) [23], glabrone (**37**, 15.5 mg) [24], kanzonol U (**38**, 21.6 mg) [11], and 8,8-dimethyl-3,4-dihydro-2*H*,8*H*pyrano[2,3-*f*]-chromon-3-ol (**39**, 17.4 mg) [25] (**Figure 4**). The structures of isolated compounds were classified into 12 groups: chalcones (**1**–**4**, **11**–**16**), isoflavans (**5**, **6**, **17**–**24**), an isoflavone (**25**), 3-arylcoumarins (**26**, **27**), pterocarpans (**28**–**30**), a flavone (**31**), a flavanol (**7**), flavanones (**32**–**35**), isoflavones (**8**, **36**, **37**), an isoflavane (**9**), an 2-aryl benzofuran (**38**), and chromones (**10**, **39**). Compounds **13**, **15**, **17**, **18**, and **36** were isolated from *G. glabra* for the first time.

Compound **1** was isolated as a yellow amorphous powder with a formula of C21H22O<sup>6</sup> determined by high-resolution electrospray ionization mass spectrometry (HRESIMS), which showed an accurate [M+H]+ ion at *m/z* 371.1487. Compound **1** had an absorption maxima at 366 and 248 nm in its UV spectrum and absorbance bands at 3427 cm−1 (hydroxy groups), 1625 cm−1 (conjugated carbonyl group), and 1595, 1507, and 1469 cm−1 (aromatic rings) in its IR spectrum. The 1 H NMR spectrum of **1** (acetone-*d*<sup>6</sup> ) contained signals for two *trans*coupled protons at δH 7.91 and 7.67 (each d, *J* = 15.7 Hz), *ortho*-coupled aromatic protons at δH 7.25 and 6.72 (each d, *J* = 8.5 Hz), *meta*-coupled aromatic protons at δH 7.55 and 7.51 (each d, *J* = 1.9 Hz), and methoxy protons at δH 3.88 (s). In addition, the 1 H NMR spectrum indicated the presence of a prenyl (3-methyl-2-butenyl) group [δH 5.40 (1H, m), 3.43 (2H, br d, *J* = 7.3 Hz), 1.77 and 1.75 (each 3H, br s)]. These data suggested that **1** was a chalcone derivative with four hydroxy groups, a methoxy group, and a prenyl group. Long-range correlations were observed in the heteronuclear multiple bond coherence (HMBC) spectrum of **1** (**Figure 5**) between H-2 (δH 7.51) and C═O (carbonyl, δC 188.0)/C-3 (δC 144.6)/C-4 (δC 148.3) Phenolics from *Glycyrrhiza glabra* and *G. uralensis* Roots and Their PPAR-γ Ligand-Binding Activity... http://dx.doi.org/10.5772/67406 81

**Figure 4.** Structures of **11**–**39** isolated from *G. glabra* roots [1].

hispaglabridin A (**23**, 13.7 mg) [14], hispaglabridin B (**24**, 10.7 mg) [14], glabrene (**25**, 28.8 mg) [16], kanzonol W (**26**, 3.0 mg) [10], glabrocoumarin (**27**, 16.4 mg) [17], shinpterocarpin (**28**, 41.0 mg) [18], *O*-methylshinpterocarpin (**29**, 32.4 mg) [18], licoagrocarpin (**30**, 6.1 mg) [19], licoflavanone A (**31**, 4.8 mg) [20], glabrol (**32**, 13.5 mg) [13], shinflavanone (**33**, 7.8 mg) [18], euchrenone a5 (**34**, 1.5 mg) [21], xambioona (**35**, 8.0 mg) [22], gancaonin L (**36**, 8.8 mg) [23], glabrone (**37**, 15.5 mg) [24], kanzonol U (**38**, 21.6 mg) [11], and 8,8-dimethyl-3,4-dihydro-2*H*,8*H*pyrano[2,3-*f*]-chromon-3-ol (**39**, 17.4 mg) [25] (**Figure 4**). The structures of isolated compounds were classified into 12 groups: chalcones (**1**–**4**, **11**–**16**), isoflavans (**5**, **6**, **17**–**24**), an isoflavone (**25**), 3-arylcoumarins (**26**, **27**), pterocarpans (**28**–**30**), a flavone (**31**), a flavanol (**7**), flavanones (**32**–**35**), isoflavones (**8**, **36**, **37**), an isoflavane (**9**), an 2-aryl benzofuran (**38**), and chromones (**10**, **39**). Compounds **13**, **15**, **17**, **18**, and **36** were isolated from *G. glabra* for the first time.

Compound **1** was isolated as a yellow amorphous powder with a formula of C21H22O<sup>6</sup>

H NMR spectrum of **1** (acetone-*d*<sup>6</sup>

d, *J* = 1.9 Hz), and methoxy protons at δH 3.88 (s). In addition, the 1

showed an accurate [M+H]+

**Figure 3.** Structures of **1–10** isolated from *G. glabra* roots [1].

80 Biological Activities and Action Mechanisms of Licorice Ingredients

its IR spectrum. The 1

mined by high-resolution electrospray ionization mass spectrometry (HRESIMS), which

at 366 and 248 nm in its UV spectrum and absorbance bands at 3427 cm−1 (hydroxy groups), 1625 cm−1 (conjugated carbonyl group), and 1595, 1507, and 1469 cm−1 (aromatic rings) in

coupled protons at δH 7.91 and 7.67 (each d, *J* = 15.7 Hz), *ortho*-coupled aromatic protons at δH 7.25 and 6.72 (each d, *J* = 8.5 Hz), *meta*-coupled aromatic protons at δH 7.55 and 7.51 (each

cated the presence of a prenyl (3-methyl-2-butenyl) group [δH 5.40 (1H, m), 3.43 (2H, br d, *J* = 7.3 Hz), 1.77 and 1.75 (each 3H, br s)]. These data suggested that **1** was a chalcone derivative with four hydroxy groups, a methoxy group, and a prenyl group. Long-range correlations were observed in the heteronuclear multiple bond coherence (HMBC) spectrum of **1** (**Figure 5**) between H-2 (δH 7.51) and C═O (carbonyl, δC 188.0)/C-3 (δC 144.6)/C-4 (δC 148.3)

ion at *m/z* 371.1487. Compound **1** had an absorption maxima

) contained signals for two *trans*-

H NMR spectrum indi-

deter-

and between H-6 (δH 7.55) and C═ O/C-1 (δC 130.7), indicating that two hydroxy groups were attached to C-3 and C-4. HMBC correlations between H-1′′ (δH 3.43) and C-4/C-6 (δC 122.8) and between H-2′′ (δH 5.40) and C-5 (δC 128.2) indicated the existence of a prenyl group at C-5. The structure of the B-ring moiety attributed to 3′,4′-dihydroxy-2′-methoxyphenyl and its linkage to C-β of the *trans*-olefinic group were determined by HMBC correlations between

**Figure 5.** Key HMBC correlations of **1**, **5**, **7**, and **8** [1].

H-5′ (δH 6.72) and C-1′ (δC 121.0)/C-3′ (δC 138.7)/C-4′ (δC 149.0), H-6′ (δH 7.25) and C-β (δC 138.4)/ C-2′ (δC 148.7), and methoxy protons and C-2′. Therefore, the structure of **1** was assigned as 3,3′,4,4′-tetrahydroxy-2′-methoxy-5-prenylchalcone.

Compound **5** was isolated as a yellow amorphous powder with a molecular formula of C21H20O<sup>5</sup> determined by HRESIMS. The 1 H NMR spectrum of **5** indicated signals characteristic of an isoflavan skeleton at δH 4.43 (ddd, *J* = 10.2, 3.4, 2.2 Hz, H-2a), 4.07 (dd, *J* = 10.2, 10.2 Hz, H-2b), 3.54 (m, H-3), 3.07 (dd, *J* = 15.5, 11.1 Hz, H-4a), and 2.90 (ddd, *J* = 15.5, 5.0, 2.2 Hz, H-4b). In addition, the spectrum of **5** indicated signals that we assigned to two aromatic protons at δ<sup>H</sup> 7.58 and 6.49 (each s), *ortho*-coupled aromatic protons at δH 6.87 and 6.32 (each d, *J* = 8.2 Hz), and a 2,2-dimethylpyran ring at δH 6.63 and 5.65 (each 1H, d, *J* = 9.8 Hz) and δH 1.40 and 1.38 (each 3H, s). Compound **5** and glabridin (**20**) have similar features in their 1 H NMR spectra. Moreover, the 1 H and 13C NMR signals at δH 9.77 and δC 195.3 indicated the presence of a formyl group, which was attached at C-5′, as determined by the HMBC correlations between the formyl proton signal and C-4′ (δC 163.6)/C-5′ (δC 115.4)/C-6′ (δC 133.9) (**Figure 5**). The circular dichroism (CD) profile of **5** was the same as that of synthetic 5′-formylglabridin prepared by formylation of **20**, indicating that the absolute configuration at C-3 was *R*. Therefore, the structure of **5** was assigned as 5′-formyl glabridin. It was notable that **5** was the first naturally occurring isoflavan with a formyl group in the B-ring portion of the compound.

Compound **7** was isolated as a yellow powder with a molecular formula of C20H20O<sup>5</sup> determined by HRESIMS. Compound **7** had an absorption maxima at 313 and 276 nm in its UV spectrum and absorption bands at 3374 cm−1 (hydroxy groups), 1673 cm−1 (a carbonyl group), and 1608, 1502, and 1463 cm−1 (aromatic rings) in its IR spectrum. The 1 H NMR spectrum of **7** showed signals that we assigned to a prenyl group at δH 5.39 (1H, m), 3.38 (2H, d, *J* = 7.3 Hz), and 1.74 and 1.72 (each 3H, br s), and two methines bearing an oxygen function at δH 5.03 and 4.59 (each d, *J* = 11.9 Hz). Furthermore, two 1,3,4-trisubstituted aromatic rings were identified from six aromatic protons comprising two ABX-type spin-coupling systems at δH 7.74 (d, *J* = 8.6 Hz), 6.64 (dd, *J* = 8.6, 2.2 Hz), and 6.41 (d, *J* = 2.2 Hz) and δH 7.35 (d, *J* = 2.0 Hz), 7.27 (dd, *J* = 8.2, 2.0 Hz), and 6.90 (d, *J* = 8.2 Hz). The above data indicated that **7** was a dihydroxyflavan-3-ol derivative with a prenyl unit. The HMBC correlations between H-5 (δH 7.74) and C-4 (δC 192.7)/C-9 (δC 164.1), H-8 (δH 6.41) and C-7 (δC 165.4)/C-9, H-6 (δH 6.64) and C-7, H-2′ (δH 7.35) and C-2 (δC 84.6)/C-4′ (δC 155.8), H-6′ (δH 7.27) and C-2/C-4′, and H-1′′ (δH 3.38) and C-2′ (δC 130.0)/C-3′ (δC 128.1)/C-4′ indicated that two hydroxy groups and a prenyl group were attached to C-7, C-4′, and C-3′, respectively (**Figure 5**). In the CD spectrum of **7**, the positive Cotton effects at 210, 240, and 334 nm and the negative Cotton effect at 304 nm indicated absolute configurations of 2*R* and 3*R* [26]. Therefore, the structure of **7** was identified as (2*R*,3*R*)-3,4′,7-trihydroxy-3′-prenylflavanone.

The following suggested that compound **8** (C21H20O<sup>5</sup> ) was an isoflavone derivative: a UV absorption maximum at λmax 263 nm [27], a proton resonance at δH 8.16 (1H, s), and a corresponding oxygen-bearing olefinic carbon signal at δC 152.2. The 1 H NMR spectrum of **8** contained signals for an aromatic proton at δH 7.54 (s), *p*-disubstituted aromatic protons at δH 7.55 and 6.98 (each d, *J* = 8.8 Hz), and methoxy protons at δ 3.84 (3H, s). In addition, the 1 H NMR spectrum implied the presence of a prenyl unit [δH 5.41 (1H, m), 3.56 (2H, d, *J* = 7.3 Hz), and 1.76 (3H × 2, br s)]. In the HMBC spectrum of **8** (**Figure 5**), correlation peaks were observed between H-5 (δH 7.54) and C-4 (δC 175.4)/C-7 (δC 148.7)/C-9 (δC 145.7)/C-1′′ (δC 28.4), methoxy protons (δH 3.84) and C-4′ (δC 159.9), and H-2 (δH 8.16) and C-8 (δC 145.7), indicating that two hydroxy groups were attached to C-7 and C-8, a methoxy group to C-4′, and a prenyl group to C-6. Therefore, the structure of **8** was found to be 7,8-dihydroxy-4′-methoxy-6-prenylisoflavanone.

In the same way, the structures of **2**–**4**, **6**, **9**, and **10** were established as shown in **Figure 3**. Compounds **3**, **4**, and **9** showed neither specific rotation nor Cotton effects in their CD spectra, indicating that these compounds were racemates.
