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.
- Glycyrrhiza glabra
- Glycyrrhiza uralensis
- metabolic syndrome
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) . 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  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
The roots of
The following suggested that compound
In the same way, the structures of
4. PPAR-γ ligand-binding activity of compounds 1–39 isolated from
5. Isolation and structural determination of phenolic compounds from
The roots of
6. PPAR-γ ligand-binding activity of compounds
40– 52isolated 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.
Fractionation of the EtOH extract of
Kuroda M, Mimaki Y, Honda S, Tanaka H, Yokota S, Mae T. Phenolics from Glycyrrhiza glabraroots and their PPAR-γ ligand-binding activity. Bioorganic & Medicinal Chemistry 2010; 18: 962–970.
Kuroda M, Mimaki Y, Sashida Y, Mae T, Kishida H, Nishiyama T, Tsukagawa M, Konishi E, Takahashi K, Kawada T, Nakagawa K, Kitahara M. Phenolics with PPAR-γ ligand-binding activity obtained from licorice ( Glycyrrhiza uralensisRoots) and ameliorative effects of glycyrin on genetically diabetic KK-Ay mice. Bioorganic & Medicinal Chemistry Letters 2003; 13: 4267–4272.
Takahashi N, Kawada T, Goto T, Yamamoto T, Taimatsu A, Matsui N, Kimura K, Saito M, Hosokawa M, Miyashita K, Fushiki T. Dual action of isoprenols on activation of both PPARγ and PPARα in 3 T3-L1 adipocytes and HepG2 hepatocytes. FEBS Letter 2002; 514: 315–322.
The samples were dissolved in DMSO, to which the medium was added to obtain the final concentration of 0.1% (v/v) of DMSO. DMSO was also added to the control wells.
Kajiyama K, Demizu S, Hiraga Y, Kinoshita K, Koyama K, Takahashi K, Tamura Y, Okada K, Kinoshita T. Two prenylated retrochalcones from Glycyrrhiza inflate.Pytochemistry 1992; 31: 3229–3232.
Saitoh T, Shibata S. New type chalcone from licorice root. Tetrahedron Letters 1975; 16: 4461–4462.
Delle Monache G, De Rosa M C, Scurria R, Vitali A, Cuteri A, Monacelli B, Pasqua G, Botta B. Comparison between metabolite productions in cell culture and in whole plant of Maclura pomifera. Phytochemistry 1995; 39: 575–580.
Kinoshita T, Kajiyama K, Hiraga Y, Takahashi K, Tamura Y, Mizutani K. The isolation of new pyrano-2-arylbenzofuran derivatives from the root of Glycyrrhiza glabra. Chemical and Pharmaceutical Bulletin 1996; 44: 1218–1221.
Ferrari F, Botta B, Alves de L R. Flavonoids and isoflavonoids from Zollernia paraensis. Phytochemistry 1983; 22: 1663–1664.
Fukai T, Sheng C B, Horikoshi T, Nomura T. Isoprenylated flavonoids from underground parts of Glycyrrhiza glabra. Phytochemistry 1996; 43: 1119–1124.
Gottlieb O.R., Braga de O.A., Goncalves T.M.M., De Oliveira G.G., Pereira S.A. Isoflavonoids from Cyclolobium species. Phytochemistry 1975; 14: 2495–2499.
Song C, Zheng Z, Liu D, Hu Z. Antimicrobial isoflavans from Astragalus membranaceus(Fisch.) Bunge. Acta Botanica Sinica 1997; 39: 486–488.
Saitoh T, Kinoshita T, Shibata S. New isoflavan and flavanone and licorice root. Chemical and Pharmaceutical Bulletin 1976; 24: 752–755.
Mitscher L A, Park Y H, Omoto S, Clark G W, Clark D. Antimicrobial agents from higher plants, Glycyrrhiza glabraL. (var. Spanish). I. Some antimicrobial isoflavans, isoflavenes, flavanones and isoflavones. Heterocycles 1978; 9: 1533–1538.
Kinoshita T, Kajiyama K, Hiraga Y, Takahashi K, Tamura Y, Mizutani K. Isoflavan derivatives from Glycyrrhiza glabra(Licorice). Heterocycles 1996; 43:581–588.
Mitscher L A, Park Y H, Clark D, Beal J L. Antimicrobial agents from higher plants. Antimicrobial isoflavanoids and related substances from Glycyrrhiza glabraL. var. typica. Journal of Natural Products 1980; 43: 259–269.
Kinoshita T, Tamura Y, Mizutani K. The isolation and structure elucidation of minor isoflavonoids from licorice of Glycyrrhiza glabraOrigin. Chemical and Pharmaceutical Bulletin 2005; 53: 847–849.
Kitagawa I, Chen W Z, Hori K, Harada E, Yasuda N, Yoshikawa M, Ren J. Chemical studies of Chinese licorice-roots. I. Elucidation of five new flavonoid constituents from the roots of Glycyrrhiza glabraL. collected in Xinjiang. Chemical and Pharmaceutical Bulletin 1994; 42: 1056-1062.
Asada Y, Li W, Yoshikawa T. Isoprenylated flavonoids from hairy root cultures of Glycyrrhiza glabra. Phytochemistry 1998; 47: 389–392.
Fukui H, Goto K, Tabata M. Two antimicrobial flavanones from the leaves of Glycyrrhiza glabra. Chemical and Pharmaceutical Bulletin 1988; 36: 4174–4176.
Mizuno M, Tamura K, Tanaka T, Iinuma M. Six flavanones from the roots of Euchresta formosana. Phytochemistry 1989; 28: 2811–2812.
Pereira M O da S, Fantine E C, De Sousa J R. Prenylated flavonoids from seeds of Calopogonium mucunoides. Phytochemistry 1982; 21; 488–489.
Fukai T, Wang Q H, Takayama M, Nomura T. Structure of five new prenylated flavonoids L, M, N, O, and P from aerial parts of Glycyrrhiza uralensis.Heterocycles 1990; 31: 373–382.
Kinoshita T, Saitoh T, Shibata S. The occurrence of an isoflavene and the corresponding isoflavone in licorice root. Chemical and Pharmaceutical Bulletin 1976; 24: 991–994.
Nomura T, Fukai T. “Phenolic constituents of licorice ( GlycyrrhizaSpecies)” in “Progress in the chemistry of organic natural products” Herz W, Kirby G W, Moore R E, Steglich W, Tamm C, Eds: Springer Wien: New York, 1998, Vol. 73, p. 27.
Gaffield W. Circular dichroism, optical rotatory dispersion and absolute configuration of flavanones, 3-hydroxyflavanones and their glycosides: Determination of aglycone chirality in flavanone. Tetrahedron 1970; 26: 4093–4108.
Kiuchi F, Chen X, Tsuda Y. Four new phenolic constituents from licorice (root of Glycyrrhizasp.). Heterocycles 1990; 31: 629–636.
Shibano M, Henmi A, Matsumoto Y, Kusano G, Miyase T, Hatakeyama Y. Studies on the index compounds for HPLC analysis of Glycyrrhiza uralensis.Heterocycles 1997; 45: 2053–2060.
Zeng L, Fukai T, Nomura T, Zhang R Y, Lou Z C. Four new prenylated flavonoids, glyasperins A, B, C, and D from the roots of Glycyrrhiza aspera. Heterocycles 1992; 34: 575–587.
Demizu S, Kajiyama K, Takahashi K, Hiraga Y, Yamamoto S, Tamura Y, Okada K, Kinoshita T. Antioxidant and antimicrobial constituents of licorice: Isolation and structure elucidation of a new benzofuran derivative. Chemical and Pharmaceutical Bulletin 1988; 36: 3474–3479.
Kinoshita T, Saitoh T, Shibata S. A new 3-arylcoumarin from licorice root. Chemical and Pharmaceutical Bulletin 1978; 26: 135–140.
Shiozawa T, Urata S, Kinoshita T, Saitoh T. Revised structures of glycyrol and isoglycyrol, constituents of the root of Glycyrrhiza uralensis. Chemical and Pharmaceutical Bulletin 1989; 37: 2239–2240.
Hatano T, Fukuda T, Miyase T, Noro T, Okuda T. Phenolic constituents of licorice. III. Structures of glicoricone and licofuranone, and inhibitory effects of licorice constituents of monoamine oxidase. Chemical and Pharmaceutical Bulletin 1991; 39: 1238–1243.
Saitoh T, Kinoshita T, Shibata S. Flavonols of licorice root. Chemical and Pharmaceutical Bulletin 1976; 24: 1242–1245.
Fukai T, Wang Q H, Kitagawa T, Kusano K, Nomura T, Iitake Y. Structures of six isoprenoid-substituted flavonoids, gancaonins F, G, H, I, glycyrol, and isoglycyrol from Xibei licorice ( GlycyrrhizaSp.). Heterocycles 1989; 29: 1761–1772.
Nakanishi T, Inada A, Kambayashi K, Yoneda K. Flavonoid glycosides of the roots of Glycyrrhiza uralensis. Phytochemistry 1985; 24: 339–341.