**6. PPAR-γ ligand-binding activity of compounds 40–52 isolated from** *G. uralensis*

Of the isolated compounds, the new compound **40** and known compounds **41**–**45** exhibited significant PPAR-γ ligand-binding activity (**Figure 6**). The activity of **40** at 5.0 μg/mL (=13.6 μM) was stronger than that of 2.0 μM TRG (relative luminescence intensity of 3.7). The coumestan derivative **46**, which was less active than **40**, was structurally similar to the active compound **43**, and the only detected difference between **43** and **46** was the formation of a five-membered ether ring between C-4 and C-2′ in **46**. This suggested that the presence of a hydroxy group at C-2′ in the isoflavan, isoflavene, or arylcoumarin skeleton is necessary for PPAR-γ ligand-binding activity. Furthermore, the isoflavones, **48** and **49**, which have a hydroxy group at C-2′ and no isoprenyl group at C-6, did not exhibit activity, suggesting that the isoprenyl group at C-6 was also involved in PPAR-γ ligand-binding activity. In conclusion, the isoprenyl group at C-6 and the C-2′ hydroxy group in the aromatic C ring of the isoflavan, isoflavene, or arylcoumarin skeleton were structural requirements for PPAR-γ ligand-binding activity (**Figure 9**).

**Figure 9.** Structural requirements for the isoflavan skeleton for PPAR-γ ligand binding [2].

#### **7. Ameliorative effects on diabetic KK-A<sup>y</sup> mice**

The ameliorative effects of glycyrin (**44**) in KK-Ay mice, an animal model of genetic type 2 diabetes, were studied using pioglitazone as a positive control. There was no difference in the food intake or body weight of mice between the treated groups and the control group. Test compound intake, calculated from the food intake and body weight of the mice, was approximately 100 mg/(kg day) in the glycyrin and glycyrol (**46**) groups and 23 mg/(kg day) in the pioglitazone group. Blood glucose levels significantly decreased after 4 days of feeding in both the glycyrin- and pioglitazone-treated groups compared to that in the control group, whereas the blood glucose levels of the glycyrol-treated group were comparable to those of the control group (**Table 1**).


a Body weights and blood glucose levels are expressed as means ± SE of five mice.

b Calculated as (total food intake) (number of mice day).

c Calculated as (average food intake/average body weight of mice).

Statistical significance is indicated as \*\* (*P* < 0.01) as determined by Dunnett's multiple comparison test.

**Table 1.** Effect of feeding glycyrin (**44**) on KK-Ay mice in experiments for the preventing diabetes [2]a .

Pioglitazone, a potent PPAR-γ agonist that activates PPAR-γ, resulted in the improvement of insulin resistance and type 2 diabetes mellitus. Glycyrin exhibited significant PPAR-γ ligandbinding activity and appeared to reduce the blood glucose levels of KK-Ay mice by the same biological mechanism as pioglitazone. This finding was supported by the observation that glycyrol, structurally related to glycyrin but lacking PPAR-γ ligand-binding activity, failed to improve the hyperglycemia of KK-Ay mice.
