Tatsumasa Mae

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.

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 AUC0–24 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.

Mechanism of action of antimycobacterial activity of the new benzoxazinorifamycin KRM-1648.

The mechanism of antimicrobial activity of KRM-1648 (KRM), a new rifamycin derivative with potent antimycobacterial activity, was studied. Both KRM and rifampin (RMP) inhibited RNA polymerases from Escherichia coli and Mycobacterium avium at low concentrations: the 50% inhibitory concentrations (IC50s) of KRM and RMP for E. coli RNA polymerase were 0.13 and 0.10 micrograms/ml, respectively, while the IC50s for M. avium RNA polymerase were 0.20 and 0.07 microgram/ml. Both KRM and RMP exerted weak inhibitory activity against Mycobacterium fortuitum RNA polymerase, rabbit thymus RNA polymerases, E. coli DNA polymerase I, and two types of reverse transcriptases. Uptake of 14C-KRM by M. avium reached 18,000 dpm/mg (dry weight) 1.5 h after incubation, while uptake by E. coli cells was slight. KRM was much more effective in inhibiting uptake of 14C-uracil than was RMP (IC50 of KRM, 0.04 microgram/ml; IC50 of RMP, 0.12 microgram/ml). These findings suggest, first, that the potent antimycobacterial activity of KRM is due to inhibition of bacterial RNA polymerase and, second, that the activity of KRM against target organisms depends on target cell wall permeability.

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-Ay 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.

Effect of a new rifamycin derivative, rifalazil, on liver microsomal enzyme induction in rat and dog

1. The effect of a new rifamycin derivative, rifalazil (KRM-1648), on liver microsomal enzyme induction was studied in rat and dog with repeated oral administration of the compound. Relative liver weight, cytochrome b5 and P450 contents, enzyme activities of NADPH-cytochrome c reductase, aniline hydroxylase, p-nitroanisole O-demethylase, aminopyrine N-demethylase, and erythromycin N-demethylase were measured. 2. In rat, rifalazil treatment at 300 mg/kg/day for 10 days increased cytochrome b5 content but it did not affect liver weight, P450 content or enzyme activities. In contrast, rifampicin and rifabutin increased relative liver weights, cytochrome contents and enzyme activities under similar conditions. 3. In dog, rifalazil did not affect any parameters at 30 or 300 mg/kg/day for 13 weeks. 4. These findings indicate that rifalazil is not an enzyme inducer in rat and dog. This property differs from other rifamycin derivatives such as rifampicin and rifabutin.

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

SUMMARY 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.

Identification of enzymes responsible for rifalazil metabolism in human liver microsomes

1. The major metabolites of rifalazil in human are 25-deacetyl-rifalazil and 32- hydroxy-rifalazil. Biotransformation to these metabolites in pooled human liver microsomes, cytosol and supernatant 9000g (S9) fractions was studied, and the enzymes responsible for rifalazil metabolism were identified using inhibitors of esterases and cytochromes P450 (CYP). 2. The 25-deacetylation and 32-hydroxylation of rifalazil occurred in incubations with microsomes or S9 but not with cytosol, indicating that both the enzymes responsible for rifalazil metabolism were microsomal. Km and Vmax

Isolation and identification of major metabolites of rifalazil in mouse and human

1. Three metabolites of rifalazil have been isolated from dog urine and identified as 25-deacetyl-rifalazil, 30-hydroxy-rifalazil and 25-deacetyl-30-hydroxy-rifalazil. In the current study major metabolites of rifalazil in mouse and human were isolated and identified, and their antimicrobial activities determined. 2. Urinary excretion of rifalazil and its metabolites in six mouse strains, CD-1 (ICR), BALB/c, C57BL/6, C3H/He, DBA/2 and CBA/J, was examined. Two major metabolites were detected in mouse urine obtained after several oral doses, and the proportion of rifalazil metabolites against total urinary excretion varied over a 2-fold range (4.8-8.7%) in the different mouse strains. 3. One of two major metabolites in mouse urine was 25-deacetyl-rifalazil and the other was unknown: it was isolated from mouse urine and identified by ms and 1H- and 13C-nmr as 32-hydroxy-rifalazil. 4. In human, two major metabolites of rifalazil were detected in urine obtained after administration of a single oral dose. These metabolites were also produced by incubation of rifalazil with pooled human liver microsomes, and identified by lc/ms and lc/ms/ms as 25-deacetyl-rifalazil and 32-hydroxy-rifalazil. 5. The antimicrobial activities of 32-hydroxy-rifalazil against gram-positive bacteria and mycobacteria were similar with those of the parent compound.

In vitro metabolism of a rifamycin derivative by animal and human liver microsomes, whole blood and expressed human CYP3A isoform.

1. In vitro metabolism of a rifamycin derivative, benzoxazinorifamycin KRM-1648, was studied using mouse, rat, guinea pig, dog, monkey and human liver microsomes. 30-Hydroxy-KRM-1648 (M2) was produced in mouse, dog, monkey and human microsomes. 25-Deacetyl-KRM-1648 (M1) was produced in dog and human microsomes, but not in mouse or monkey microsomes. Neither M1 nor M2 was detected in rat or guinea pig microsomes. 2. In dog and human liver microsomes the formation of M2 was dependent on NADPH, but the formation of M1 was not. 3. In vitro metabolism of the parent compound was studied in whole blood in some species. Only M1 was detected in mouse and rat blood, and not in dog and human blood. 4. These findings demonstrated that the metabolite pattern in dog resembled that in man, and suggested that the 30-hydroxylation of KRM-1648 was mediated by cytochrome P450, but that the 25-deacetylation was not. 5. Among the ten recombinant human P450 isoforms used, only the cell lysates including CYP3A3 and CYP3A4 catalysed the M2 formation from KRM-1648.