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Ligand-Binding Activity Affects Diabetes in KK-Ay Mice, Abdominal Obesity in Diet-Induced Obese C57BL Mice and Hypertension in Spontaneously Hypertensive Rats
Functional Foods Development Division and
* Life Science Research Laboratories, Life Science RD Center, Kaneka Corporation, Takasago, Hyogo 676-8688, Japan;
Laboratory of Medicinal Plant Science, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji 192-0392, Japan;
** Department of Molecular Metabolism and Diabetes, Department of Internal Medicine, Tohoku University School of Medicine, Sendai 980-8574, Japan; and
Laboratory of Nutrition Chemistry, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
1To whom correspondence should be addressed. E-mail: Tatsumasa.Mae{at}kn.kaneka.co.jp.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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(PPAR-) ligands such as thiazolidinediones are effective against this syndrome. In this study, we showed that nonaqueous fractions of licorice (Glycyrrhiza uralensis Fisher) extracted with ethanol, ethyl acetate and acetone, but not an aqueous extract, had PPAR- ligand-binding activity with a GAL4-PPAR- chimera assay. Some prenylflavonoids including glycycoumarin, glycyrin, dehydroglyasperin C and dehydroglyasperin D, a newly found compound, were identified as active compounds with PPAR- ligand-binding activity in the nonaqueous fraction of licorice. A licorice ethanolic extract contained these four active compounds at a total concentration of 16.7 g/100 g extract. Feeding the licorice ethanolic extract at 0.1–0.3 g/100 g diet [
100 to 300 mg/(kg body · d)] for 4 wk decreased (P < 0.05) blood glucose level in younger (6 wk old) and older (13 wk old) diabetic KK-Ay mice and reduced (P < 0.05) weights of intra-abdominal adipose tissues in high fat diet–induced obese C57BL mice. An increase in blood pressure in spontaneously hypertensive rats was suppressed (P < 0.01) by 3 wk of oral administration of the licorice ethanolic extract at 300 mg/(kg body · d). These findings indicate that licorice ethanolic extract is effective in preventing and ameliorating diabetes, ameliorating abdominal obesity and preventing hypertension, and suggest that licorice ethanolic extract would be effective in preventing and/or ameliorating the metabolic syndrome.
KEY WORDS: • licorice extract • prenylflavonoids • PPAR- ligand • metabolic syndrome
Type 2 diabetes, obesity/abdominal obesity, hypertension and dyslipidemia are closely linked with insulin resistance; clustering of these risk factors in the same person has been called the metabolic syndrome, which is a major public health problem (1,2). Adipocytes are highly specialized cells that play critical roles in energy regulation and homeostasis. Adipocyte differentiation is a tightly controlled process in which determinant genes such as peroxisome proliferator-activated receptor- (PPAR-)1 and CCAAT/enhancer binding protein-
lead to programmed adipose cell differentiation (3,4).
PPAR are ligand-activated transcription factors belonging to the nuclear receptor superfamily. There are three PPAR subtypes; they are the products of distinct genes and are commonly designated PPAR-, PPAR- and PPAR-
(5,6). PPAR- is the predominant molecular target for insulin-sensitizing thiazolidinedione drugs such as troglitazone, pioglitazone and rosiglitazone, which have been approved for use in type 2 diabetic patients (7,8). A link between PPAR- and diseases including dyslipidemia, hypertension, inflammation, atherosclerosis and cancer has been suggested (5,9,10). Many studies of thiazolidinediones have indicated that PPAR- agonists are effective against the metabolic syndrome or insulin resistance syndrome (11–13). Therefore, many chemical compounds have been developed as PPAR- agonists.
On the other hand, natural PPAR- ligands were unknown until PUFA such as linoleic acid, linolenic acid, arachidonic acid and eicosapentaenoic acid demonstrated PPAR- ligand-binding activity, although they also bind to other PPAR subtypes (14,15). Recently, it was reported that some flavonoids (16,17), isoprenols (18) and triterpene acids (19) can activate PPAR-. This finding suggests that some edible plants have PPAR- agonistic activity and can be used as functional foods and natural medicines to prevent and ameliorate the metabolic syndrome.
Therefore, to discover a novel natural PPAR- ligand in edible plants, we tested extracts of 70 edible plants such as spices and herbs, and found that an extract of licorice (Glycyrrhiza uralensis Fisher) had higher PPAR- ligand-binding activity than extracts of the other plants tested. Licorice, the roots of the leguminous plant Glycyrrhiza species, has been used for >4000 years since ancient Egyptian times, and is one of the most frequently employed botanicals in traditional medicine (20).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Troglitazone purified from Noscal tablets (Sankyo, Tokyo, Japan) was used in an in vitro PPAR- ligand-binding assay. Enalapril maleate from Hashima Laboratory, Nihon Bioresearch (Hashima, Japan) was used in an experiment for the prevention of hypertension. Other chemicals, solvents and reagents were commercially available.
Preparation of licorice extracts.
The roots of licorice harvested in the northwestern region of China (Seihoku-kanzo), Glycyrrhiza uralensis Fischer, were extracted with 5 volumes of ethanol, ethyl acetate or acetone, and the extract was obtained by filtering and evaporating the solvent. An aqueous extract of licorice was prepared by extraction with 20 volumes of water (80°C), filtration and lyophilization.
PPAR- ligand-binding assay.
PPAR- ligand-binding activity was assayed in a GAL4-PPAR- chimera assay system (18). In this system, luciferase expression from a reporter plasmid through transcriptional activation by a fusion protein of GAL4 DNA-binding domain (amino acids 1–147) and human PPAR- ligand-binding domain (167–467) bound to a compound with PPAR- ligand-binding activity was measured. A compound that could bind to the fusion protein but not transactive it tested negative in this assay.
CV-1 monkey kidney cells from the American Type Culture Collection (Manassas, VA) were grown in DMEM-supplemented 100 mL/L fetal bovine serum (FBS) in a 37°C/5% CO2 incubator. Two plasmids, pM-hPPAR- and p4xUASg-tk-luc, were transfected into cells cultured in 96-well plates using LipofectAMINE Plus (Invitrogen Japan, Tokyo, Japan) in test wells. In mock control wells, pM and p4xUASg-tk-luc were transfected. pM-hPPAR- is a plasmid expressing a fusion protein of GAL4 DNA-binding domain and human PPAR- ligand-binding domain, and p4xUASg-tk-luc is a reporter plasmid containing four copies of a 17-mer upstream activating sequence for GAL4 DNA-binding domain and thymidine kinase gene promoter (tk-promoter) in front of luciferase cDNA. pM is a plasmid containing the GAL4 DNA-binding domain but not human PPAR- ligand-binding domain of pM-hPPAR-. Plasmid construction was described in the report by Takahashi et al. (18). After 24 h of transfection, the medium was changed to DMEM-supplemented 100 mL/L charcoal-treated FBS and sample, and the cells were cultured for 24 h. The sample was dissolved in dimethyl sulfoxide (DMSO), and added to the medium at a final concentration of 1 mL/L of DMSO. DMSO was used as a solvent control and troglitazone at 0.5–2 µmol/L as a positive control. All samples were assayed in four wells each in an experiment. The cells were washed twice with PBS containing Ca++ and Mg++, and luciferase activities were measured using LucLite (PerkinElmer Life Science Japan K.K., Tokyo, Japan) in a TopCount plate-reader (PerkinElmer Life Science Japan). Relative luciferase activity of a sample was calculated as a ratio of mean luciferase activity in the test wells to that in the mock control wells, and PPAR- ligand-binding activity is presented as a ratio of relative luciferase activity of a sample to that of the solvent control.
PPAR- ligand-binding activity at 0.5 µmol/L of troglitazone was approximately twice that of the solvent control. When the activity of a tested sample was more than twofold the control, the sample was judged to have PPAR- ligand-binding activity.
HPLC analysis.
Analysis was performed using a J’sphere octadecylsilane (ODS)-H80 column, 4.6 x 250 mm (YMC, Kyoto, Japan) at 40°C. The mobile phase was a gradient of 10 mmol/L phosphoric acid (solvent A) and acetonitrile (solvent B) at a flow rate of 1 mL/min with 35% B for 0–15 min, linear increase from 35 to 70% B for 15–65 min, 70% B for 65–70 min and 35% B for 70–90 min. Peaks were detected at a wavelength of 254 nm. The injection volume of a sample was 20 µL in methanol solution.
Identification of compounds.
The licorice extract obtained with ethyl acetate was separated into four fractions by silica-gel column chromatography eluted with chloroform/methanol (19:1 and 9:1, v/v), chloroform/methanol/water (4:1:0.1, v/v/v) and methanol. Twenty-four compounds from the chloroform/methanol (19:1, v/v) fraction and two compounds from the chloroform/methanol/water fraction were isolated by silica gel column chromatography, ODS-silica gel column chromatography, HPLC with an ODS column, gel filtration column chromatography and preparative TLC. Their structures were identified by 1H- and 13C-NMR.
Animal experiments.
Female genetically diabetic KK-Ay/Ta mice (Clea Japan, Tokyo, Japan) were used in experiments for prevention and amelioration of diabetes, female C57BL/6J mice (Clea Japan) in an experiment for amelioration of abdominal obesity and male spontaneously hypertensive rats (SHR/N; Charles River Japan, Yokohama, Japan) in an experiment for prevention of hypertension. Animals were housed in an animal laboratory controlled environment at 20–24°C temperature, 45–65% humidity and 12-h (0730–1930 h) light:dark cycle for mice, and at 20–26°C temperature, 40–70% humidity and 12-h (0600–1800 h) light:dark cycle for rats. Mice were reared with their respective groups in a cage, and rats individually.
Experiments for prevention and amelioration of diabetes.
The effect of licorice ethanolic extract on diabetes in female KK-Ay mice was examined in a preventive experiment and an ameliorative experiment. In the preventive experiment, 6-wk-old mice were divided into three groups of five mice each by body weight and blood glucose level, and fed purified powdered diet (Table 1; Oriental Yeast, Tokyo, Japan) or purified powdered diet containing licorice ethanolic extract at 0.1 or 0.2 g/100 g diet. In the preventive experiment, the 13-wk-old mice were divided into three groups of six mice each, and fed powdered CE-2 diet (Clea Japan,) or powdered CE-2 diet containing licorice ethanolic extract at 0.1 or 0.3 g/100 g diet. Mice consumed these diets and water ad libitum for 4 wk in both experiments. Blood glucose levels measured from the tail vein were determined using a blood glucose level monitor Glutest Ace (Sanwa Kagaku Kenkyusho, Nagoya, Japan) before and after 4 wk of feeding. Then the mice were killed to collect blood and periuterine, perirenal and mesenteric adipose tissues. Serum insulin levels were measured using an Insulin Measurement Kit (Morinaga Institute of Biological Science, Yokohama, Japan). The collected adipose tissues were pooled as intra-abdominal adipose tissue and its relative weight to body weight was measured.
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Male C57BL mice (8 wk old) were fed a high fat diet (Table 1; Oriental Yeast) for 8 wk to induce obesity, and were divided into three groups of seven mice each by body weight. The mice in the control group were fed powdered CE-2 diet, and the mice in the licorice groups, powdered CE-2 diet containing licorice ethanolic extract at 0.1 or 0.2 g/100g diet. Mice consumed these diets and water ad libitum for 4 wk. Then the mice were killed, and perirenal, periuterine and mesenteric adipose tissues were collected and their relative weights to body weight were measured.
Experiment for prevention of hypertension.
The preventive effect of licorice ethanolic extract on hypertension in male SHR was examined in Hashima Laboratory, Nihon Bioresearch. Rats (5 wk old) were divided into three groups of eight rats each by body weight and blood pressure level. The rats in the licorice group were orally administered licorice ethanolic extract suspended in propylene glycol at a daily dose of 300 mg/(kg body · d) for 3 wk; rats in a control group were administered vehicle, 3 mL/(kg body · d) of propylene glycol. As a positive control, enalapril maleate in 5 g/L methyl cellulose was orally administered at 20 mg/(kg body · d) over the same period. Rats consumed CRF-1 diet (Oriental Yeast) and water ad libitum. Systolic blood pressure levels determined from the tail artery were measured weekly using a BP-98A blood pressure monitor (Softlon, Tokyo, Japan).
Statistical analysis.
Data were analyzed using the SAS/STAT software computerized statistical analysis program (SAS Institute, Carey, NC). When significant differences were indicated by one-way ANOVA, Tukey’s multiple comparison test was applied. Differences were considered significant at P < 0.05. Values in the text are mean ± SEM.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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ligand-binding activity of licorice extract.
Licorice extract was one of the positive samples with higher PPAR- ligand-binding activity than other samples tested in the screening of extracts of 70 edible plants such as spices and herbs with a GAL4-PPAR- chimera assay. The activities of nonaqueous fractions of licorice extracted with ethanol, ethyl acetate and acetone were similar, but an aqueous extract of licorice had no activity (Fig. 1). Yields of these extracts obtained with ethanol, ethyl acetate, acetone and water were 14.8, 8.2, 11.0 and 21.0 g/100 g licorice, respectively. The activities at 30 mg/L of the nonaqueous extracts of licorice were equivalent to that at 1 µmol/L (0.44 mg/L) of troglitazone, a synthetic potent PPAR- agonist.
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ligands in licorice extract.
Many peaks were detected in the chromatogram of licorice ethanolic extract by HPLC analysis (Fig. 2A). Chromatograms of the licorice extracts obtained with ethyl acetate and acetone were similar to that of the licorice ethanolic extract. We therefore separated the licorice extract obtained with ethyl acetate into four fractions by silica-gel column chromatography. PPAR- ligand-binding activity was detected in the chloroform/methanol (19:1, v/v) fraction, but not in the chloroform/methanol/water (4:1:0.1, v/v/v) fraction. We further isolated 24 compounds from the active fraction and two compounds from the inactive fraction, and identified their structures. Almost all of the main peaks in the chromatogram of licorice ethanolic extract (Fig. 2A) were identified and determined to be the 20 compounds listed in Table 2. Of these compounds, one was a newly found compound identified as 3-(2',4'-dihydroxyphenyl)-6–3'',3''-dimethylallyl)-5,7-dimethoxy-2H-chromene, which we named "dehydroglyasperin D." Two compounds from the inactive fraction were identified as liquiritin and isoliquiritin. Six other compounds not listed in Table 2, which were detected as minor or inseparable peaks in the chromatogram, were echinatin (a chalcone), glyasperin B (an isoflavanone), glyasperin D (an isoflavan), kaempferol 3-O-methylester, licoflavonol and topazolin (flavonols). Notably, many of the isolated compounds were prenylflavonoids, which have one or two plenyl groups with a flavonoid structure. On the other hand, a major peak detected in the chromatogram of aqueous extract of licorice (Fig. 2B) was glycyrrhizin, whose concentration was >6 g/100 g extract. Almost none of the licorice flavonoids were detected in the aqueous extract of licorice (Fig. 2B). Glycyrrhizin concentration in the licorice ethanolic extract was <0.4 g/100 g extract.
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chimera assay. When compounds were tested at 5 mg/L (12–15 µmol/L), active compounds included 3-arylcoumarins, isoflav-3-enes, isoflavanones, isoflavones, flavonols and chalcones, but flavanones, coumestans and 2-arylbenzofurans had less activity (Table 2). Six other compounds not listed in Table 2 also had PPAR- ligand-binding activity (data not shown). However, glycyrrhizin and its metabolite glycyrrhetinic acid, which were obtained as commercial reagents, exhibited no activity when tested at 2–10 mg/L (2.4–12 and 4.2–21 µmol/L, respectively) (data not shown). Among active compounds, concentrations of glycycoumarin, glycyrin, dehydroglyasperin C and dehydroglyasperin D were >2 g/100 g in the licorice ethanolic extract (Table 2), and the sum of concentrations of these four compounds was 16.7 g/100 g extract. The structures of these four compounds were similar (Fig. 3A), but the PPAR- ligand-binding activities of isoflave-3-ens, dehydroglyasperin C and dehydroglyasperin D were higher than those of 3-arylcoumarins, glycycoumarin and glycyrin (Figs. 3B, C). The concentration exhibiting activity equivalent to that of 1 µmol/L of troglitazone was 1–2 mg/L for isoflave-3-ens and 5 mg/L for 3-arylcoumarins.
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In the preventive experiment, 4 wk feeding of licorice ethanolic extract at 0.1 and 0.2 g/100 g diet did not affect body weight gain or food intake (Table 3). Intakes of licorice ethanolic extract calculated from total food intake and mean body weight of the mice were 152 and 285 mg/(kg body · d) at 0.1 and 0.2 g/100 g diet feeding, respectively (Table 3), and the doses of licorice ethanolic extract corresponded to 25 and 48 mg/(kg body · d) of the total of the four active compounds, i.e., glycycoumarin, glycyrin, dehydroglyasperin C and dehydroglyasperin D. The blood glucose level in the control was 8.2 ± 0.5 mmol/L before feeding and 28.0 ± 1.5 mmol/L after 4 wk of feeding, indicating that the mice were becoming hyperglycemic. Compared with the control, the blood glucose level was lower (P < 0.01) in mice fed licorice ethanolic extract at 0.1 and 0.2 g/100 g diet for 4 wk (Table 3), indicating a suppression of the increase in blood glucose level. Serum insulin levels in the licorice ethanolic extract groups were also lower (P < 0.05) than in the control (Table 3). Furthermore, the relative weight of intra-abdominal adipose tissue was lower (P < 0.05) in mice fed licorice ethanolic extract at 0.2 g/100 g diet than in the control (Table 3). Licorice ethanolic extract thus reduced hyperglycemia, hyperinsulinemia and abdominal fat accumulation.
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Experiment for amelioration of abdominal obesity.
Weights of periuterine, perirenal and mesenteric adipose tissues in mice fed a high fat diet for 8 wk were approximately twice those in mice fed a normal diet (data not shown). After the diet-induced obese mice were fed for 4 wk, body weight gains in the control and licorice ethanolic extract 0.1 and 0.2 g/100 g diet groups were -1.1 ± 0.3, -2.0 ± 0.5 and -1.6 ± 0.5 g/4 wk, respectively, and food intakes were 3.3, 3.5, and 3.4 g/(mouse · d), respectively. Therefore, body weight gain and food intake in the licorice ethanolic extract groups did not differ from control values. Intakes of licorice ethanolic extract were 148 and 276 mg/(kg body · d) at 0.1 and 0.2 g/100 g diet feeding, respectively, and the doses of licorice ethanolic extract corresponded to 25 and 46 mg/(kg body · d) of the total of the four active compounds, i.e., glycycoumarin, glycyrin, dehydroglyasperin C and dehydroglyasperin D. Relative weights of periuterine and perirenal adipose tissues of the mice fed licorice ethanolic extract were lower (P < 0.05), and relative weight of mesenteric adipose tissue tended to be lower (P = 0.063 and 0.089 at 0.1 and 0.2 g/100 g diet, respectively), than in the control (Fig. 4). In addition, the relative weight of intra-abdominal adipose tissue, which was the sum of periuterine, perirenal and mesenteric adipose tissues, was also lower (P < 0.05) with feeding of licorice ethanolic extract than in the control. Licorice ethanolic extract thus reduced abdominal obesity induced by high-fat diet.
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When vehicle, licorice ethanolic extract at 300 mg/(kg body · d) and enalapril maleate at 20 mg/(kg body · d) were administered orally to SHR for 3 wk, body weight gain during the experimental period did not differ among the groups. The dose of licorice ethanolic extract corresponded to 50 mg/(kg body · d) of the total of the four active compounds, i.e., glycycoumarin, glycyrin, dehydroglyasperin C and dehydroglyasperin D. The blood pressure level in the vehicle control increased dependently of time (P < 0.01) in wk 1–3 (Fig. 5), indicating that rats were developing hypertension. In rats administered enalapril maleate, an angiotensin converting enzyme inhibitor, the blood pressure levels at wk 1–3 were lower (P < 0.01) than in the vehicle control (Fig. 5). Enalapril maleate strongly suppressed an increase in blood pressure. With administration of licorice ethanolic extract, the blood pressure level tended to be lower at wk 1 (P = 0.071) and wk 2 (P = 0.091), and was lower at wk 3 (P < 0.01) than in the vehicle control (Fig. 5). Licorice ethanolic extract thus reduced hypertension.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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ligands. Moreover, we demonstrated effects of licorice ethanolic extract on diabetes, abdominal obesity and hypertension using animal models.
PPAR- ligand-binding activity was detected only in the nonaqueous fractions of licorice extracted with organic solvents such as ethanol, ethyl acetate and acetone, and not in the aqueous extract (Fig. 1). Major components of licorice include glycyrrhizin (glycyrrhizinic acid) and many phenolic compounds such as flavonoids (22). HPLC analysis indicated that the nonaqueous extract of licorice contained many flavonoids and less glycyrrhizin (<0.4 g/100 g extract), whereas the aqueous extract was rich in glycyrrhizin (>6 g/100 g extract) and poor in flavonoids (Fig. 2). Neither glycyrrhizin nor glycyrrhetinic acid had PPAR- ligand-binding activity (data not shown). These findings indicated that some flavonoids contained in the nonaqueous extract of licorice had PPAR- ligand-binding activity. It would be interesting to determine whether not only G. uralensis but other species of licorice such as G. glabra and G. inflata have PPAR- ligand-binding activity because some of the flavonoids in licorice are species specific (20).
In general, PPAR- agonists exhibit comparable potency and efficacy in assays using either the GAL4-PPAR- chimera or the full-length receptor (5). In addition, PPAR- agonists inhibit production of monocyte inflammatory cytokines such as tumor necrosis factor (TNF)-, interleukin (IL)-6 and IL-1ß (23). In another study, we examined the effect of licorice ethanolic extract on TNF- production induced by phorbol myristyl acetate (PMA) in human peripheral blood mononuclear cells prepared by plastic adherence from whole-blood buffy-coat fractions. Licorice ethanolic extract at 3–30 mg/L dose dependently inhibited TNF- production induced by PMA, but the aqueous extract of licorice at 30 mg/L and glycyrrhizin at 10 mg/L (12 µmol/L) did not (data not shown). These findings suggest that active compounds with PPAR- ligand-binding activity in the nonaqueous extract of licorice are natural PPAR- agonists.
Approximately 300 kinds of phenolic compounds have been isolated from various species of licorice, and were documented in a review article (22). In every species of licorice, 40–70 kinds of flavonoids and other phenolic compounds occur, with some species specificity in chemical structure (20); prenylflavonoids are peculiar to licorice (22). Among isolated flavonoids, four compounds, glycycoumarin, glycyrin, dehydroglyasperin C and dehydroglyasperin D, were the most predominant compounds with PPAR- ligand-binding activity in licorice ethanolic extract (Table 2, Fig. 3). When these four compounds were mixed at the same concentrations as in licorice ethanolic extract, PPAR- ligand-binding activity of the mixture was equivalent to 90% of that of licorice ethanolic extract (data not shown). Accordingly, these four compounds were the most important PPAR- ligands in licorice ethanolic extract. Dehydroglyasperin D is a newly discovered compound, and few reports are available concerning glycycoumarin, glycyrin and dehydroglyasperin C. For glycycoumarin and glycyrin, inhibition of xanthine oxidase and monoamine oxidase (24) and antibacterial activities against methicillin-resistant Staphylococcus aureus (25), upper airway respiratory tract bacteria (26) and Helicobacter pylori (27) have been reported. This is the first report on the PPAR- ligand-binding activity of prenylflavonoids.
To demonstrate the effect of licorice ethanolic extract on type 2 diabetes, genetically diabetic KK-Ay mice were used. The preventive effect was examined in not yet hyperglycemic younger (6 wk old) mice and the ameliorative effect was examined in older (13 wk old) mice with hyperglycemia. In both experiments, licorice ethanolic extract at 0.1–0.3 g/100 g diet did not affect body weight gain or food intake, although it was used at doses corresponding to 100 to 300 mg/(kg body · d). A hypoglycemic effect of licorice ethanolic extract was observed in both experiments, indicating that the extract was effective in preventing and ameliorating hyperglycemia. However, differences between the experiments were observed in serum insulin level and relative weight of intra-abdominal adipose tissue. In the control group, mean serum insulin levels were 17.7 and 26.7 nmol/L at 10 and 17 wk, respectively, in aged mice (Table 3). It appeared in the ameliorative experiment that serum insulin level could be returned to close to the initial level with a higher dose of licorice ethanolic extract. For the difference in the relative weight of intra-abdominal adipose tissue, it is conceivable that licorice ethanolic extract could inhibit accumulation of abdominal fat in a phase in which body weight increased and diabetes developed in KK-Ay mice, but could not reduce abdominal fat accumulated after body weight had increased and diabetes had developed.
Subsequently, diet-induced obese C57BL mice were used to demonstrate the effect of licorice ethanolic extract on abdominal obesity, because C57BL mice gradually become obese with feeding of a high-fat diet (28,29); however, KK-Ay mice become seriously obese for genetic reasons. In the diet-induced obese mice, licorice ethanolic extract at 0.1 to 0.2 g/100 g diet, corresponding to 148 to 276 mg/(kg body · d), significantly decreased relative weights of intra-abdominal adipose tissues (Fig. 4), indicating reduction of accumulated abdominal fat. In preliminary microscopic observations, the size of adipocytes in intra-abdominal adipose tissue was smaller than that in the control (data not shown). The PPAR- agonist thiazolidinediones improve body fat distribution in human patients, i.e., although subcutaneous fat accumulation is increased, visceral fat accumulation and the ratio of visceral/subcutaneous fat accumulation are decreased by treatment with troglitazone (30–35), pioglitazone (36) and rosiglitazone (37). Similar to the thiazolidinediones, licorice ethanolic extract should also inhibit accumulation of visceral fat and reduce accumulated visceral fat; however, clinical study is required to confirm this.
We also demonstrated that licorice ethanolic extract at 300 mg/(kg body · d) postponed the onset of hypertension in a model of spontaneous hypertension (Fig. 5). Thiazolidinediones, which are PPAR- agonists, decrease blood pressure in various models of hypertension such as fructose- and diet-fed rats (38), Zucker fatty rats (39), SHR (40,41), angiotensin II-infused rats (42), and deoxycorticosterone acetate-salt rats (43). PPAR- is expressed in various rat tissues, including blood vessels, heart, muscle, kidney, liver and adipose tissue, and PPAR- levels in aorta and mesenteric arteries are greater in SHR than in control Wistar-Kyoto rats (44). Hence, our results suggest that the blood pressure–lowering effect of licorice ethanolic extract involves a mechanism similar to that of thiazolidinediones. Interestingly, glycyrrhizin is metabolized to glycyrrhetinic acid and causes hypertension at high doses or with long-term consumption (45–47). Daily consumption of 50 g of licorice, corresponding to a daily intake of 75 mg glycyrrhetinic acid, by healthy volunteers for 2 wk significantly increased blood pressure (47). Therefore, although consumption of licorice and its aqueous extract induces hypertension, it can be expected that licorice ethanolic extract containing less glycyrrhizin would have a preventive effect on hypertension.
Licorice has been used for foods and traditional medicines for >4000 years since ancient Egyptian times (20). Licorice and its aqueous extract are used as flavoring and sweetening agents in food products because they contain glycyrrhizin as a major component. Glycyrrhizin and its metabolite glycyrrhetinic acid have many pharmacologic and physiologic effects, e.g., mineral corticoid-like activity, anti-inflammatory, antiulcer, anticarcinogenetic, antiallergy, antiviral and hepatoprotective effects (20,48,49). We showed that licorice ethanolic extract had PPAR- ligand-binding activity, and its active compounds were identified. The yield of licorice ethanolic extract was 14.8 g/100 g licorice, and total concentration of the four active compounds was 16.7 g/100 g extract. Licorice ethanolic extract decreased blood glucose level, reduced relative weight of abdominal fat, and suppressed an increase in blood pressure in animal models at doses of 100–300 mg/(kg body · d) corresponding to 17–50 mg/(kg body · d) of the four active compounds. In the diabetic and abdominal obese models, the dose-response relationship for licorice ethanolic extract was weak and not obvious between 100 and 300 mg/(kg body · d). Further study of a wider range of doses will be required to clarify the dose-response characteristics of licorice ethanol extract. In addition, pharmacokinetic study will be necessary to elucidate the absorption, distribution, metabolism and elimination of the four active compounds in licorice ethanolic extract. The metabolic syndrome is a cluster of type 2 diabetes, obesity/abdominal obesity, hypertension and dyslipidemia (1,2). Our findings using animal models suggest that licorice ethanolic extract may prevent and ameliorate the metabolic syndrome, which is becoming a serious public health problem in contemporary society.
The spectral data of a newly found compound, dehydroglyasperin D [3-(2',4'-dihydroxyphenyl)-6–3'',3''-dimethylallyl)-5,7-dimethoxy-2H-chromene], are as follows: electron impact-MS m/z, 368.1610 [M]+ (C22H24O5); UV 
max in methanol (nm), 330 (log
= 4.25) and 243 sh (log = 4.21); infrared in KBr (cm-1), 3375, 2929, 1613, 1516, 1458, 1308, 1198, 1164, 1114, 1092, 1024, 978 and 837; 1H NMR in DMSO-d6 (ppm), 7.06 (1H, d, J = 8.4 Hz, H-6'), 6.68 (1H, s, H-4), 6.34 (1H, d, J = 2.3 Hz, H-3'), 6.33 (1H, s, H-8), 6.26 (1H, dd, J = 8.4, 2.3 Hz, H-5'), 5.09 (1H, br t, J = 6.9 Hz, H-10), 4.90 (2H, s, H-2), 3.75 (3H, s, C-7-OMe), 3.67 (3H, s, C-5-OMe), 3.18 (2H, br d, J = 6.7 Hz, H-9), 1.71 (3H, s, Me-13) and 1.63 (3H, s, Me-12); 13C NMR in DMSO-d6 (ppm), 67.9 (C-2), 128.8 (C-3), 114.7 (C-4), 110.4 (C-4a), 154.9 (C-5), 115.6 (C-6), 158.0 (C-7), 95.8 (C-8), 153.2 (C-8a), 22.6 (C-9), 124.1 (C-10), 130.4 (C-11), 26.0 (C-12), 18.1 (C-13), 116.8 (C-1'), 156.7 (C-2'), 103.3 (C-3'), 158.7 (C-4'), 107.4 (C-5'), 129.2 (C-6'), 62.3 (C-5-OMe) and 56.3 (C-7-OMe).
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FOOTNOTES |
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, peroxisome proliferator-activated receptor-; SHR, spontaneously hypertensive rats; TNF, tumor necrosis factor. 
Manuscript received 4 June 2003. Initial review completed 9 July 2003. Revision accepted 2 September 2003.
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