Hypoglycemic effects of clove (Syzygium aromaticum flower buds) on genetically diabetic KK-Ay mice and identification of the active ingredients.

Clove (Syzygium aromaticum flower buds) EtOH extract significantly suppressed an increase in blood glucose level in type 2 diabetic KK-A(y) mice. In-vitro evaluation showed the extract had human peroxisome proliferator-activated receptor (PPAR)-γ ligand-binding activity in a GAL4-PPAR-γ chimera assay. Bioassay-guided fractionation of the EtOH extract resulted in the isolation of eight compounds, of which dehydrodieugenol (2) and dehydrodieugenol B (3) had potent PPAR-γ ligand-binding activities, whereas oleanolic acid (4), a major constituent in the EtOH extract, had moderate activity. Furthermore, 2 and 3 were shown to stimulate 3T3-L1 preadipocyte differentiation through PPAR-γ activation. These results indicate that clove has potential as a functional food ingredient for the prevention of type 2 diabetes and that 2-4 mainly contribute to its hypoglycemic effects via PPAR-γ activation.

Phenolics from Glycyrrhiza glabra roots and their PPAR-gamma ligand-binding activity.

Bioassay-guided fractionation of the EtOH extract of licorice (Glycyrrhiza glabra roots), using a GAL-4-PPAR-gamma chimera assay method, resulted in the isolation of 39 phenolics, including 10 new compounds (1-10). The structures of the new compounds were determined by analysis of their spectroscopic data. Among the isolated compounds, 5'-formylglabridin (5), (2R,3R)-3,4',7-trihydroxy-3'-prenylflavane (7), echinatin, (3R)-2',3',7-trihydroxy-4'-methoxyisoflavan, kanzonol X, kanzonol W, shinpterocarpin, licoflavanone A, glabrol, shinflavanone, gancaonin L, and glabrone all exhibited significant PPAR-gamma ligand-binding activity. The activity of these compounds at a sample concentration of 10microg/mL was three times more potent than that of 0.5microM troglitazone.

Licorice flavonoid oil reduces total body fat and visceral fat in overweight subjects: A randomized, double-blind, placebo-controlled study.

OBJECTIVES: To evaluate effects of licorice flavonoid oil (LFO) on total body fat and visceral fat together with body weight, body mass index (BMI) and safety parameters in overweight subjects. METHODS: In this randomized, double-blind, placebo-controlled study, moderately overweight participants (56 males, 28 females, BMI 24-30 kg/m(2)) were assigned to four groups receiving a daily dose of either 0 (placebo), 300, 600, or 900 mg of LFO. Total body fat mass was measured by dual-energy X-ray absorptiometry (DXA) and visceral fat area by abdominal computed tomography (CT) scan at baseline and after 8 weeks of LFO ingestion. Body weight, BMI, and blood samples were examined at baseline and after 4 and 8 weeks of LFO ingestion. RESULTS: Although caloric intake was similar in all four groups, total body fat mass decreased significantly in the three LFO groups after 8 weeks of ingestion. LFO (900 mg/day) resulted in significant decreases from baseline levels in visceral fat area, body weight, BMI, and LDL-cholesterol. No significant adverse effects were observed.

Clinical safety of licorice flavonoid oil (LFO) and pharmacokinetics of glabridin in healthy humans.

OBJECTIVE: Licorice flavonoids have various physiological activities such as abdominal fat-lowering, hypoglycemic and antioxidant effects. Licorice flavonoid oil (LFO: Kaneka Glavonoid Rich Oil) is a new dietary ingredient containing licorice flavonoids dissolved in medium-chain triglycerides (MCT). Glabridin is one of the bioactive flavonoids included specifically in licorice Glycyrrhiza glabra L. and is the most abundant flavonoid in LFO. In this study, we assessed the safety of LFO in healthy humans and determined the plasma concentration profile of glabridin as a marker compound. METHODS: A single-dose and two multiple-dose studies at low (300 mg), moderate (600 mg) and high (1200 mg) daily doses of LFO were carried out using a placebo-controlled single-blind design. In each study the safety of LFO and the pharmacokinetics of glabridin were assessed. RESULTS: Pharmacokinetic analysis in the single-dose study with healthy male subjects (n = 5) showed that glabridin was absorbed and reached the maximum concentration (Cmax) after approximately 4 h (Tmax), and then eliminated relatively slowly in a single phase with a T1/2 of approximately 10 h at all doses. The Cmax and AUC(0-24 h) increased almost linearly with dose. The multiple-dose studies with healthy male and female subjects for 1 week and 4 weeks suggested that plasma glabridin reached steady state levels within 2 weeks with a single daily administration of 300 to 1200 mg/day LFO. In these human studies at three dose levels, there were no clinically noteworthy changes in hematological or related biochemical parameters. All clinical events observed were mild and considered to be unrelated to LFO administration even after repeated administration for 4 weeks. CONCLUSION: These studies demonstrated that LFO is safe when administered once daily up to 1200 mg/day. This is the first report on the safety of licorice flavonoids in an oil preparation and the first report on the pharmacokinetics of glabridin in human subjects.

Suppression by licorice flavonoids of abdominal fat accumulation and body weight gain in high-fat diet-induced obese C57BL/6J mice.

We applied licorice flavonoid oil (LFO) to high-fat diet-induced obese C57BL/6J mice and investigated its effect. LFO contains hydrophobic flavonoids obtained from licorice by extraction with ethanol. The oil is a mixture of medium-chain triglycerides, having glabridin, a major flavonoid of licorice, concentrated to 1.2% (w/w). Obese mice were fed on a high-fat diet containing LFO at 0 (control), 0.5%, 1.0%, or 2.0% for 8 weeks. Compared with mice in the control group, those in the 1% and 2% LFO groups efficiently reduced the weight of abdominal white adipose tissues and body weight gain. A histological examination revealed that the adipocytes became smaller and the fatty degenerative state of the hepatocytes was improved in the 2% LFO group. A DNA microarray analysis of the liver showed up-regulation of those genes for beta-oxidation and down-regulation of those for fatty acid synthesis in the 2% LFO group. These findings suggest that LFO prevented and ameliorated diet-induced obesity via the regulation of lipid metabolism-related gene expression in the liver.

Effects of ingested turmeric oleoresin on glucose and lipid metabolisms in obese diabetic mice: a DNA microarray study.

Turmeric, the rhizome of Curcuma longa L., has a wide range of effects on human health. Turmeric oleoresin, an extract of turmeric, is often used for flavoring and coloring. Curcuminoids and turmeric essential oil are both contained in turmeric oleoresin, and both of these fractions have hypoglycemic effects. In the present study, we comprehensively assessed the effect of turmeric oleoresin on hepatic gene expression in obese diabetic KK-Ay mice using DNA microarray analysis and quantitative real-time polymerase chain reaction (PCR). Female KK-Ay mice aged 6 weeks (n = 6/group) were fed a high-fat diet containing turmeric oleoresin, curcuminoids, and essential oil for 5 weeks. The same diet without any of these fractions was used as a control diet. Ingestion of turmeric oleoresin and essential oil inhibited the development of increased blood glucose and abdominal fat mass, while curcuminoids only inhibited the increase in blood glucose. DNA microarray analysis indicated that turmeric oleoresin ingestion up-regulated the expression of genes related to glycolysis, beta-oxidation, and cholesterol metabolism in the liver of KK-Ay mice, while expression of gluconeogenesis-related genes was down-regulated. Real-time PCR analysis was conducted to assess the contribution of the curcuminoids and essential oil in turmeric oleoresin to the changes in expression of representative genes selected by DNA microarray analysis. This analysis suggested that curcuminoids regulated turmeric oleoresin ingestion-induced expression of glycolysis-related genes and also that curcuminoids and turmeric essential oil acted synergistically to regulate the peroxisomal beta-oxidation-related gene expression induced by turmeric oleoresin ingestion. These changes in gene expression were considered to be the mechanism by which the turmeric oleoresin affected the control of both blood glucose levels and abdominal adipose tissue masses. All of these results suggest that the use of whole turmeric oleoresin is more effective than the use of either curcuminoids or the essential oil alone.

Determination of glabridin in human plasma by solid-phase extraction and LC-MS/MS.

Glabridin is a major flavonoid included specifically in licorice (Glycyrrhiza glabra L.), and has various physiological activities including antioxidant and anti-inflammatory effects. We have developed and validated an analytical method for determination of glabridin in human plasma by solid-phase extraction (SPE) and LC-MS/MS. Glabridin was extracted from plasma by SPE using a C8 cartridge and analyzed by LC-MS/MS using mefenamic acid as an internal standard (IS). The analyte were separated by a C18 column on LC, and monitored with a fragment ion of m/z 201 formed from a molecular ion of m/z 323 for glabridin and that of m/z 196 from m/z 240 for IS during negative ion mode with tandem MS detection. The lower limit of quantitation (LLOQ) of glabridin was 0.1 ng/mL in plasma, corresponding to 1.25 pg injected on-column. The calibration curves exhibited excellent linearity (r>0.997) between 0.1 and 50 ng/mL. Precision and accuracy were <17 and <+/-7% at LLOQ, and <11 and <+/-5% at other concentrations. Glabridin was recovered >90%, and was stable when kept at 10 degrees C for 72 h, at -20 degrees C until 12 weeks, and after three freeze-thaw cycles. This is the first report on determination of glabridin in body fluids by the selective, sensitive, and reproducible method.

Hypoglycemic effects of turmeric (Curcuma longa L. rhizomes) on genetically diabetic KK-Ay mice.

The turmeric (Curcuma longa L. rhizomes) EtOH extract significantly suppressed an increase in blood glucose level in type 2 diabetic KK-A(y) mice. In an in vitro evaluation, the extract stimulated human adipocyte differentiation in a dose-dependent manner and showed human peroxisome proliferator-activated receptor (PPAR)-gamma ligand-binding activity in a GAL4-PPAR-gamma chimera assay. The main constituents of the extract were identified as curcumin, demethoxycurcumin, bisdemethoxycurcumin, and ar-turmerone, which had also PPAR-gamma ligand-binding activity. These results indicate that turmeric is a promising ingredient of functional food for the prevention and/or amelioration of type 2 diabetes and that curcumin, demethoxycurcumin, bisdemethoxycurcumin, and ar-turmerone mainly contribute to the effects via PPAR-gamma activation.

Curcuminoids and sesquiterpenoids in turmeric (Curcuma longa L.) suppress an increase in blood glucose level in type 2 diabetic KK-Ay mice.

Turmeric, the rhizome of Curcuma longa L., has a wide range of effects on human health. The chemistry includes curcuminoids and sesquiterpenoids as components, which are known to have antioxidative, anticarcinogenic, and antiinflammatory activities. In this study, we investigated the effects of three turmeric extracts on blood glucose levels in type 2 diabetic KK-A(y) mice (6 weeks old, n = 5/group). These turmeric extracts were obtained by ethanol extraction (E-ext) to yield both curcuminoids and sesquiterpenoids, hexane extraction (H-ext) to yield sesquiterpenoids, and ethanol extraction from hexane-extraction residue (HE-ext) to yield curcuminoids. The control group was fed a basal diet, while the other groups were fed a diet containing 0.1 or 0.5 g of H-ext or HE-ext/100 g of diet or 0.2 or 1.0 g of E-ext/100 g of diet for 4 weeks. Although blood glucose levels in the control group significantly increased (P < 0.01) after 4 weeks, feeding of 0.2 or 1.0 g of E-ext, 0.5 g of H-ext, and 0.5 g of HE-ext/100 g of diet suppressed the significant increase in blood glucose levels. Furthermore, E-ext stimulated human adipocyte differentiation, and these turmeric extracts had human peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligand-binding activity in a GAL4-PPAR-gamma chimera assay. Also, curcumin, demethoxycurcumin, bisdemethoxycurcumin, and ar-turmerone had PPAR-gamma ligand-binding activity. These results indicate that both curcuminoids and sesquiterpenoids in turmeric exhibit hypoglycemic effects via PPAR-gamma activation as one of the mechanisms, and suggest that E-ext including curcuminoids and sesquiterpenoids has the additive or synergistic effects of both components.

Licorice flavonoids suppress abdominal fat accumulation and increase in blood glucose level in obese diabetic KK-A(y) mice.

Licorice, the root of the Glycyrrhiza species, is one of the most frequently employed botanicals in traditional medicines. In this study, we investigated the effects of hydrophobic flavonoids from Glycyrrhiza glabra LINNE on abdominal fat accumulation and blood glucose level in obese diabetic KK-A(y) mice. In order to enrich a fraction of hydrophobic flavonoids, licorice flavonoid oil (LFO) was prepared by further extracting licorice ethanolic extract with medium-chain triglycerides (MCT), and adjusting the concentration of glabridin, the major flavonoid of licorice, to 1.2% in oil. KK-A(y) mice aged 6 weeks were assigned to 5 groups (n=6 each), and fed a high-fat diet containing 0 (control), 0.5%, 1%, or 2% LFO, or 0.5% conjugated linoleic acid (CLA) for 4 weeks. Compared with the control, body weight gain and weights of abdominal adipose tissues were suppressed (p<0.05) by feeding the diet containing 2% LFO, and blood glucose levels after 2 and 4 weeks were suppressed by all of the diets containing LFO. Although CLA feeding suppressed (p<0.05) body weight gain, it increased (p<0.05) blood glucose level after 2 weeks compared with the control level. Furthermore, LFO and licorice ethanolic extract stimulated human adipocyte differentiation in vitro. These results indicate that licorice hydrophobic flavonoids have abdominal fat-lowering and hypoglycemic effects, possibly mediated via activation of peroxisome proliferator-activated receptor-gamma (PPAR-gamma).

Regulation of interleukin-8 secretion in human intestinal epithelial Caco-2 cells by alpha-humulene.

It is well known that various cytokines such as interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-alpha are expressed and secreted from intestinal epithelial cells and that these cytokines affect the immune cells beneath the intestinal epithelial monolayers. As the secretion of these cytokines is likely to be regulated by food-derived substances, we focused on those food substances which regulate the secretion of IL-8 in human intestinal epithelial Caco-2 cells. 72 food samples extracted with 40% ethanol were tested, and the extracts of peppermint and dokudami significantly increased the IL-8 secretion. Among the compounds known to be contained in peppermint and dokudami, alpha-humulene substantially increased the IL-8 secretion.alpha-Humulene had no significant effect on the secretion of such other soluble factors as TNF-alpha, IL-1beta, IL-6, or NGF, suggesting that the effect of alpha-humulene was specific for IL-8 secretion. The expression level of IL-8 mRNA was significantly increased by treating with alpha-humulene. These results suggest that the secretion of IL-8 by alpha-humulene is regulated at the transcriptional level.

Phenolics with PPAR-gamma ligand-binding activity obtained from licorice (Glycyrrhiza uralensis roots) and ameliorative effects of glycyrin on genetically diabetic KK-A(y) mice.

The EtOAc extract of licorice (Glycyrrhiza uralensis roots) exhibited considerable PPAR-gamma ligand-binding activity. Bioassay-guided fractionation of the extract using a GAL-4-PPAR-gamma chimera assay method resulted in the isolation of two isoflavenes, one of which is a new compound named dehydroglyasperin D, an isoflavan, two 3-arylcoumarins, and an isoflavanone as the PPAR-gamma ligand-binding active ingredients of licorice. The isoprenyl group at C-6 and the C-2' hydroxyl group in the aromatic ring-C part in the isoflavan, isoflavene, or arylcoumarin skeleton were found to be the structural requirements for PPAR-gamma ligand-binding activity. Glycyrin, one of the main PPAR-gamma ligands of licorice, significantly decreased the blood glucose levels of genetically diabetic KK-A(y) mice.

A licorice ethanolic extract with peroxisome proliferator-activated receptor-gamma ligand-binding activity affects diabetes in KK-Ay mice, abdominal obesity in diet-induced obese C57BL mice and hypertension in spontaneously hypertensive rats.

The metabolic syndrome, including type 2 diabetes, insulin resistance, obesity/abdominal obesity, hypertension and dyslipidemia, is a major public health problem. Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) 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-gamma ligand-binding activity with a GAL4-PPAR-gamma chimera assay. Some prenylflavonoids including glycycoumarin, glycyrin, dehydroglyasperin C and dehydroglyasperin D, a newly found compound, were identified as active compounds with PPAR-gamma 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 [approximately 100 to 300 mg/(kg body x 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 x 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.