Effect of feeding glycyrin (
Abstract
The EtOH extract of Glycyrrhiza glabra roots and the EtOAc extract of Glycyrrhiza uralensis roots exhibited considerable PPAR-γ ligand-binding activity. Bioassay-guided fractionation of these extracts resulted in the isolation of 52 phenolics, including 11 novel ones. The PPAR-γ ligand-binding activity of more than 10 isolated phenolics at 10 μg/mL was approximately three times greater than that of 0.5 μM triglitazone. Glycyrin (44), isolated from the EtOAc extract of G. uralensis roots as a PPAR-γ ligand, reduced the blood glucose levels of genetically diabetic KK-Ay mice through its PPAR-γ ligand-binding activity.
Keywords
- phenolics
- Glycyrrhiza glabra
- Glycyrrhiza uralensis
- PPAR-γ
- metabolic syndrome
1. Introduction
Peroxisome proliferator-activated receptor (PPAR)-γ is the primary molecular target for insulin-sensitizing thiazolidinedione drugs. These drugs activate PPAR-γ, increasing the number of small adipocytes that differentiate from preadipocytes and inducing apoptosis in large adipocytes. Because small adipocytes function normally, whereas large adipocytes hyperproduce and hypersecrete adipocytokines, an increased ratio of small adipocytes to large adipocytes improves insulin resistance. Therefore, compounds with PPAR-γ ligand-binding activity may be useful for the prevention and improvement of type 2 diabetes, a representative insulin resistance syndrome. We found that the EtOH extract of
In this chapter, we describe the results of the bioassay-guided fractionation of
2. PPAR-γ ligand-binding activity
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 ascorbic acid. The cells were then inoculated into a 96-well culture plate at 6 × 103 cells/well and incubated in 5% CO2/air at 37°C for 24 h. Cells were washed with OPTI-minimum essential 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) determined for each sample.
3. Isolation and structural determination of phenolic compounds from G. glabra
The roots of
Compound
Compound
Compound
The following suggested that compound
In the same way, the structures of
4. PPAR-γ ligand-binding activity of compounds 1–39 isolated from G. glabra
Compounds
5. Isolation and structural determination of phenolic compounds from G. uralensis
The roots of
6. PPAR-γ ligand-binding activity of compounds 40 –52 isolated from G. uralensis
Of the isolated compounds, the new compound
7. Ameliorative effects on diabetic KK-Ay mice
The ameliorative effects of glycyrin (
Control | Glycyrin (0.10%) | Glycyrol (0.10%) | Pioglitazone (0.02%) | |
---|---|---|---|---|
Body weight (g) | ||||
Day 0 | 52.6 ± 0.53 | 54.1 ± 1.78 | 52.6 ± 1.07 | 55.1 ± 0.69 |
Day 4 | 48.9 ± 0.48 | 50.4 ± 1.58 | 49.1 ± 1.02 | 53.6 ± 1.07 |
Day 7 | 50.4 ± 0.56 | 51.9 ± 1.62 | 50.3 ± 0.91 | 55.8 ± 1.50 |
Day 10 | 46.9 ± 0.42 | 48.1 ± 1.57 | 46.8 ± 0.08 | 52.5 ± 11.41 |
Average food intake (g/mouse/day)b | 5.43 | 5.34 | 5.47 | 6.34 |
Average test compound intake (mg/kg/day)c | 0 | 102 | 108 | 23 |
Blood glucose level (mg/dL) | ||||
Day 0 | 476 ± 22 | 474 ± 27 | 427 ± 24 | 486 ± 26 |
Day 4 | 420 ± 14 | 278 ± 14** | 421 ± 19 | 191 ± 6** |
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 ligand-binding 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.
8. Conclusion
Fractionation of the EtOH extract of
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