Kaku Nakagawa

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.

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.

Genotoxicity studies on licorice flavonoid oil (LFO)

Licorice flavonoid oil (LFO) is a new functional food ingredient. In this study, the genotoxicity of LFO was investigated using a test battery of three different methods. In a reverse mutation assay using four Salmonella typhimurium strains and Escherichia coli, LFO did not increase the number of revertant colonies in any tester strain with or without metabolic activation by rat liver S9 mix. In a chromosomal aberration test using Chinese hamster lung (CHL/IU) cells, LFO did not induce any chromosomal aberrations either in the short period test without rat liver S9 mix or in the continuous treatment (24 h or 48 h) test. However, in the short-period test with rat liver S9 mix, LFO induced structural chromosomal aberrations at concentrations higher than 0.6 mg/mL. A bone marrow micronucleus test using male F344 rats was initially conducted. The animals were dosed by oral gavage at doses up to 5000 mg/kg/day. No significant or dose-dependent increases in the frequency of micronucleated polychromatic erythrocytes (MNPCE) were observed and the high dose suppressed the ratio of polychromatic erythrocytes (PCE) to total erythrocytes. Subsequently, a liver and peripheral blood micronucleus test using male F344 rats was conducted. No micronuclei induction either in hepatocytes or PCE was observed even at the highest dose of 5000 mg/kg/day. From the findings obtained from the genotoxicity assays performed in this study and the published pharmacokinetic studies of LFO, it appears unlikely that dietary consumption of LFO will present any genotoxic hazard to humans.

90-Day repeated-dose toxicity study of licorice flavonoid oil (LFO) in rats

Licorice flavonoid oil (LFO) is a new functional food ingredient consisting of licorice hydrophobic polyphenols in medium-chain triglycerides (MCT). As part of a safety evaluation, a 90-day oral toxicity study in rats was conducted using an LFO concentrate solution (2.90% glabridin). Male and female animals were assigned to one of 12 groups (10 males or females per group) and received corn oil (negative control), MCT (vehicle control), or 400, 600, 800 or 1600 mg/kg of the LFO concentrate solution. In conclusion, LFO concentrate solution induced an anticoagulation effect in both sexes, although there was a clear sex difference. Based on these findings, it is concluded that the no-observed-adverse-effect level (NOAEL) for the LFO concentrate solution is estimated to be 800 mg/kg/day for female rats, and approximately 400 mg/kg/day for male rats.

Glabridin induces glucose uptake via the AMP-activated protein kinase pathway in muscle cells

The present study demonstrates that glabridin, a prenylated isoflavone in licorice, stimulates glucose uptake through the adenosine monophosphate-activated protein kinase (AMPK) pathway in L6 myotubes. Treatment with glabridin for 4h induced glucose uptake in a dose-dependent manner accompanied by the translocation of glucose transporter type 4 (GLUT4) to the plasma membrane. Glabridin needed at least 4h to increase glucose uptake, while it significantly decreased glycogen and increased lactic acid within 15 min. Pharmacological inhibition of AMPK by Compound C suppressed the glabridin-induced glucose uptake, whereas phosphoinositide 3-kinase and Akt inhibition by LY294002 and Akt1/2 inhibitor, respectively, did not. Furthermore, glabridin induced AMPK phosphorylation, and siRNA for AMPK completely abolished glabridin-induced glucose uptake. We confirmed that glabridin-rich licorice extract prevent glucose intolerance accompanied by the AMPK-dependent GLUT4 translocation in the plasma membrane of mice skeletal muscle. These results indicate that glabridin may possess a therapeutic effect on metabolic disorders, such as diabetes and hyperglycemia, by modulating glucose metabolism through AMPK in skeletal muscle cells.

Inhibition by licorice flavonoid oil of glutathione S-transferase-positive foci in the medium-term rat hepatocarcinogenesis bioassay.

Licorice flavonoid oil (LFO) is a new functional food ingredient consisting of hydrophobic licorice polyphenols in medium-chain triglycerides. Recently, it was reported that licorice and its derivatives have anticarcinogenic activity in some types of tumors. However, the anticarcinogenic activity has not been identified in the liver, which is a major target organ for carcinogenesis in human. Therefore, we hypothesized that LFO has antihepatocarcinogenic activity, and we tested this hypothesis using the rat medium-term liver bioassay for carcinogens. Six-week-old male F344 rats (15 animals/group) received N-diethylnitrosamine (200 mg/kg by intraperitoneal injection) to initiate carcinogenesis. From the second week after initiation, animals received a 6-week regimen of either LFO concentrate solution (0, 150, 300, or 600 mg/kg) intragastrically or phenobarbital sodium salt in the diet (500 ppm) as a positive control. During the third week after initiation, animals were subjected to a two-thirds partial hepatectomy. During the eighth week of the treatment period, liver samples were taken from animals and examined immunohistochemically for expression of glutathione S-transferase placental form. No increase in the number of glutathione S-transferase placental form-positive liver foci was observed in all LFO groups compared with the negative control (solvent) group, and the number of foci in the 600 mg/kg LFO group was significantly lower than that in the negative control group. These results indicate that LFO concentrate solution has a significant inhibitory effect on liver carcinogenesis at 600 mg/kg.

Enhancement of Fat Oxidation by Licorice Flavonoid Oil in Healthy Humans during Light Exercise

Licorice flavonoid oil (LFO) is a new functional food ingredient consisting of hydrophobic licorice polyphenols in medium-chain triglycerides. Recent studies reported that LFO prevented and ameliorated diet-induced obesity via the regulation of lipid metabolism-related gene expression in the livers of mice and rats, while it reduced body weight in overweight human subjects by reducing total body fat. However, the direct effects of LFO on energy metabolism have not been studied in human subjects. Therefore, we investigated the effects of ingestion of LFO on energy metabolism, including fat oxidation, by measuring body surface temperature under resting conditions and respiratory gas analysis under exercise conditions in healthy humans. We showed that ingestion of a single 600 mg dose of LFO elevated body trunk skin temperature when measured in a slightly cooled air-conditioned room, and increased oxygen consumption and decreased the respiratory exchange ratio as measured by respiratory gas analysis during 40% Vo2max exercise with a cycle ergometer. Furthermore, repeated ingestion of 300 mg of LFO for 8 d decreased respiratory exchange during the recovery period following 40 min of 30% Vo2max exercise on a treadmill. These results suggest that LFO enhances fat oxidation in humans during light exercise.

Licorice flavonoid oil enhances muscle mass in KK- A y mice

Aims: Muscle mass is regulated by the balance between the synthesis and degradation of muscle proteins. Loss of skeletal muscle mass is associated with an increased risk of developing metabolic diseases such as obesity and type 2 diabetes mellitus. The aim of this study was to clarify the effects of licorice flavonoid oil on muscle mass in KK‐Ay/Ta mice. Main methods: Male genetically type II diabetic KK‐Ay/Ta mice received 0, 1, or 1.5 g/kg BW of licorice flavonoid oil by mouth once daily for 4 weeks. After 4 weeks, the femoral and soleus muscles were collected for western blotting for evaluation of the mTOR/p70 S6K, p38/FoxO3a, and Akt/FoxO3a signaling pathways. Key findings: Ingestion of licorice flavonoid oil significantly enhanced femoral muscle mass without affecting body weight in KK‐Ay/Ta mice. Licorice flavonoid oil also decreased expression of MuRF1 and atrogin‐1, which are both markers of muscle atrophy. The mechanisms by which licorice flavonoid oil enhances muscle mass include activation of mTOR and p70 S6K, and regulation of phosphorylation of FoxO3a. Significance: Ingestion of licorice flavonoids may help to prevent muscle atrophy.

Effects of Pediococcus acidilactici R037 on Serum Triglyceride Levels in Mice and Rats after Oral Administration

The biological effects of heat-killed Pediococcus acidilactici R037 (R037) were evaluated when orally administered in mice and rats. Oral R037 administration at a daily dose of 10 and 100 mg/kg for 3 wk dose-dependently reduced fasting and non-fasting serum triglyceride concentrations in KK-Ay/TaJcl mice, a model of type II diabetes, obesity, hypercholesterolemia, and hypertriglyceridemia. Serum levels of free fatty acids in the 100 mg/kg group tended to decrease (not statistically significant), and total cholesterol levels remained unchanged. Treatment with R037 resulted in a significant decrease in blood glucose (at 100 mg/kg) and liver weight (at 10 and 100 mg/kg), and a small body weight gain (at 100 mg/kg) as compared to those in control mice. In addition, oral R037 administration at 100, 200, and 400 mg/kg/d for 1 wk dose-dependently suppressed the increase in serum triglyceride levels in Wistar rats after oral fat loading. Moreover, intraduodenal injection of 120 mg of R037 in Wistar rats suppressed gastric vagal nerve activity (GVNA) indicating suppression of intestinal digestion and absorption of food, and suppression of appetite. The R037 injection potentiated epididymal white adipose tissue sympathetic nerve activity (WAT-SNA) and tended to potentiate pancreatic sympathetic nerve activity (PSNA), suggesting that R037 activated lipolysis. Taken together, these findings indicate that R037 lowers serum triglycerides, possibly through suppressing intestinal absorption and potentiating lipolytic pathways. R037 may be useful for primary prevention of coronary artery diseases in subjects with mild or borderline dyslipidemia in combination with lifestyle changes.