In Vitro Production of Optically Active Octahydrocurcumin by Human Intestinal Bacterium.

Enterococcus avium, producing 5R-hexahydrocurcumin metabolized tetrahydrocurcumin to octahydrocurcumin in vitro. Based on a detailed analysis of the two secondary alcohols, the metabolite obtained from tetrahydrocurcumin via 5R-hexahydrocurcumin was identified as 3R,5R-octahydrocurcumin. The activities of 5R-hexahydrocurcumin and 3R,5R-octahydrocurcumin were compared to those of the synthetic compounds, using monocyte chemoattractant protein-1 produced via murine adipocytes in vitro. The optically active curcuminoids reduced the cytokine production similar to tetrahydrocurcumin without any difference in their stereochemistry.

Effects of isoflavone-rich red clover extract on blood glucose level: A randomized, double-blind, placebo-controlled trial.

High blood glucose is associated with increased risk of various diseases. Red clover (RC; Trifolium pratense L.) is an edible legume whose sprout is rich in isoflavones such as formononetin and biochanin A. We examined the effects of RC extract on postprandial and fasting blood glucose level, using a randomized, double-blind, placebo-controlled trial with 36 participants, aged 25 to 64 years, who were randomly assigned to receive either 1.91 g of RC extract (containing 8 mg formononetin and 1.8 mg biochanin A) or placebo. Each participant ingested the assigned test food daily for 8 weeks, and at the oral maltose tolerance test (OMTT). Initially, the two groups did not significantly differ in OMTT results. However, fasting insulin levels at 8 weeks were significantly lower in the RC group (4.76 µIU/ml at Week 0 to 4.01 µIU/ml at Week 8) with a significant interaction (P = 0.046). Subgroup analysis showed that change in blood glucose level (blood glucose ΔC) tended to decrease late in the trial period during OMTT in the ≤50-year-old RC group, as did fasting blood glucose and insulin levels at 8 weeks; hemoglobin A1c was also significantly reduced in this subgroup (5.36% at Week 0 to 5.28% at Week 8) with a significant interaction (P = 0.040). These results suggest that the daily intake of RC could reduce blood glucose, particularly for those ≤50 years old. Formononetin-an α-glucosidase inhibitor-is considered to be the major functional molecule for these effects. Therefore, intake of RC that contains formononetin might help blood glucose control.

Production of Optically Active Hexahydrocurcumin by Human Intestinal Bacterium in Vitro.

A hexahydrocurcumin-producing bacterium named 2a1-2b was isolated from human feces. It was observed that the bacterium had more than 99% similarity with Enterococcus avium ATCC14025T according to 16S ribosomal DNA (rDNA) sequence. The strain 2a1-2b produced optically active 5R-hexahydrocurcumin (enantiomeric excess (e.e.) > 95%) from tetrahydrocurcumin but not from curcumin. Our results showed that intestine is an important place for producing hexahydrocurcumin.

Benzyl Isothiocyanate Produced by Garden Cress (Lepidium sativum) Prevents Accumulation of Hepatic Lipids.

We determined the physiological effects of glucotropaeolin-rich lyophilized garden cress sprout powder (GC) administered to fasting and nonfasting mice. High-performance liquid chromatography analysis revealed glucotropaeolin (57.4±1.1 mg/g dry weight) as a major phytochemical constituent of GC. Decreasing tendency in body weight and feeding efficiency ratio were detected in the group of mice fed 0.05% (w/w) GC (GC0.05). Nonfasting mice exhibited significantly lower liver weights that were unchanged after fasting. Decreased total lipid (TL) and triglyceride (TG) levels in the liver were detected in the nonfasted GC0.01 and GC0.05 groups, but only in TLs of the fasted groups. The levels of plasma TGs and nonesterified fatty acids of the GC0.05 group, which remained unchanged during nonfasting, decreased after fasting. To determine its effects on the accumulation of lipids in the liver, the glucotropaeolin aglycone, benzyl isothiocyanate (BITC), was added to the liver-derived HepG2 human cell line cultured in a medium containing a high concentration of D-glucose (4,500 mg/L D-glucose) (HG group) or 1 mM oleic acid (SO group). Toxicity was not detected when cells were treated with as much as 5 µM BITC; however, lipid accumulation was inhibited by BITC in a concentration-dependent manner in the HG groups. The same effect was observed when 2 µM BITC was added to the diet of the SO groups. These results suggest that moderate levels of GC or BITC are useful for reducing liver and plasma TGs.

Equol Inhibits Mushroom Tyrosinase in Vitro through Tight Binding.

Equol, an intestinal metabolite of daidzein, inhibited more potently mushroom tyrosinase in vitro than other inhibitors, genistein and kojic acid. We investigated the mechanism underlying tyrosinase inhibition by equol. Treating racemic equol with tyrosinase produced 3'-hydroxyequol. Because the optical activity of the product showed <25% enantiomeric excess, the reaction was not highly stereospecific. Using enzyme-linked immunosorbent assays with an anti-equol monoclonal antibody, we observed that equol bound to pre-coated tyrosinase in a dose-dependent manner. Our results suggested the formation of a stable equol-tyrosinase complex.

Red Clover (Trifolium pratense L.) Sprout Prevents Metabolic Syndrome.

We examined the prevention effect of red clover (Trifolium pratense L.) sprout on metabolic syndrome using a high-carbohydrate and high-fat diet (Western diet; WD)-induced male C57BL/6J obese model mouse. Red clover sprout-lyophilized powder (RC) contained 3.5 mg/g dry-weight of formononetin as a major phenolic compound, as analyzed by high performance liquid chromatography. Supplementation of 0.3% (w/w) RC in a WD (WD+RC) showed an anti-obesity effect and ameliorated lipid metabolism in the obese model mice. Additionally, fasting plasma glucose levels were significantly reduced in the WD+RC group. Administration of 0.1 mg/kg formononetin reduced the postprandial blood glucose level, as assessed using the oral maltose tolerance test. However, no significant formononetin intake effect was observed on the plasma insulin level. These results suggest that the formononetin contained in red clover sprout inhibits α-glucosidase and thereby contributes to reducing the postprandial blood glucose response in mice.

Daidzein reductase of Eggerthella sp. YY7918, its octameric subunit structure containing FMN/FAD/4Fe-4S, and its enantioselective production of R-dihydroisoflavones.

S-Equol is a metabolite of daidzein, a type of soy isoflavone, and three reductases are involved in the conversion of daidzein by specific intestinal bacteria. S-Equol is thought to prevent hormone-dependent diseases. We previously identified the equol producing gene cluster (eqlABC) of Eggerthella sp. YY7918. Daidzein reductase (DZNR), encoded by eqlA, catalyzes the reduction of daidzein to dihydrodaidzein (the first step of equol synthesis), which was confirmed using a recombinant enzyme produced in Escherichia coli. Here, we purified recombinant DZNR to homogeneity and analyzed its enzymological properties. DZNR contained FMN, FAD, and one 4Fe-4S cluster per 70-kDa subunit as enzymatic cofactors. DZNR reduced the CC bond between the C-2 and C-3 positions of daidzein, genistein, glycitein, and formononetin in the presence of NADPH. R-Dihydrodaidzein and R-dihydrogenistein were highly stereo-selectively produced from daidzein and genistein. The Km and kcat for daidzein were 11.9 µM and 6.7 s-1, and these values for genistein were 74.1 µM and 28.3 s-1, respectively. This enzyme showed similar kinetic parameters and wide substrate specificity for isoflavone molecules. Thus, this enzyme appears to be an isoflavone reductase. Gel filtration chromatography and chemical cross-linking analysis of the active form of DZNR suggested that the enzyme consists of an octameric subunit structure. We confirmed this by small-angle X-ray scattering and transmission electron microscopy at a magnification of ×200,000. DZNR formed a globular four-petal cloverleaf structure with a central vertical hole. The maximum particle size was 173 Å.

Th17 Cell Induction by Adhesion of Microbes to Intestinal Epithelial Cells.

Intestinal Th17 cells are induced and accumulate in response to colonization with a subgroup of intestinal microbes such as segmented filamentous bacteria (SFB) and certain extracellular pathogens. Here, we show that adhesion of microbes to intestinal epithelial cells (ECs) is a critical cue for Th17 induction. Upon monocolonization of germ-free mice or rats with SFB indigenous to mice (M-SFB) or rats (R-SFB), M-SFB and R-SFB showed host-specific adhesion to small intestinal ECs, accompanied by host-specific induction of Th17 cells. Citrobacter rodentium and Escherichia coli O157 triggered similar Th17 responses, whereas adhesion-defective mutants of these microbes failed to do so. Moreover, a mixture of 20 bacterial strains, which were selected and isolated from fecal samples of a patient with ulcerative colitis on the basis of their ability to cause a robust induction of Th17 cells in the mouse colon, also exhibited EC-adhesive characteristics.

Epidemiology of anorexia nervosa in Japanese adolescents.

BACKGROUND: No epidemiologic survey examining eating disorders in Japan has been done at a national level since 1992. The prevalence of anorexia nervosa, as assessed by questionnaires to hospitals, is thought to be underestimated because patients with anorexia nervosa tend to avoid consultations. In conformity with the School Health and Safety Act of Japan, schools are required to have physicians perform a medical examination of students every year. The teachers in charge of health education and school physicians determine the height, weight, and health condition, and examine the medical records of each student. Therefore, we as members of the Survey Committee for Eating Disorders of the Japanese Ministry of Health, Labour, and Welfare conducted an epidemiologic survey using questionnaires sent to schools in seven prefectures to determine the current prevalence of anorexia nervosa among adolescents. METHODS: We sent a questionnaire to elementary, junior high, and senior high schools. Questionnaires contained items on the number of students, patients with anorexia nervosa in each grade who were diagnosed by specialists, and students who the school physician strongly suspected to have anorexia nervosa but who did not undergo a clinical examination in a medical institution. RESULTS: We found patients of both sexes with anorexia nervosa aged 9-10 years in elementary schools. The point prevalence of anorexia nervosa for girls, including strongly suspected cases, in the three grades of junior high school and three grades of senior high school were 0-0.17 %, 0-0.21 %, 0.17-0.40 %, 0.05-0.56 %, 0.17-0.42 % and 0.09-0.43 %, respectively. We also confirmed a prominent sex difference in the prevalence of anorexia nervosa. The prevalence of boys was one third that of girls in some prefectures. One third to one half of diagnosed and strongly suspected students with anorexia nervosa had not received medical consultation or treatment. CONCLUSIONS: Although the prevalence of anorexia nervosa had regional differences in Japan, it has reached levels comparable to those in Western societies. Because no eating disorder center exists and the treatment environment is poor, national action to address this disease is a pressing need in Japan.

Stereochemical determination of O-desmethylangolensin produced from daidzein.

We had isolated an O-desmethylangolensin (O-DMA)-producing bacterium, Clostridium rRNA cluster XIVa strain SY8519. According to chiral separation using HPLC, the SY8519-produced O-DMA exhibited high optical purity. To determine the absolute stereochemistry of O-DMA, we prepared 2-(4-hydroxyphenyl)propionic acid (2-HPPA) from the O-DMA using the Baeyer-Villiger reaction. From chiral analysis of the product, the major peak had the same stereochemistry to that of 2-HPPA produced from genistein by the same bacteria. As we have determined the stereochemistry of SY8519-produced 2-HPPA to have an R configuration, by the chemical synthesis of (S)-2-HPPA, the SY8519-produced O-DMA must also possess R stereochemistry at the 2-position. To study the stereoselective metabolism, we applied racemic dihydrodaidzein to SY8519. The O-DMA was isolated from the culture media and starting material was also recovered. The O-DMA produced was optically active in a similar manner to that produced from daidzein. However, the remaining dihydrodaidzein exhibited no difference between the enantiomers. These results suggested that SY8519 produces (R)-O-DMA from both enantiomers of dihydrodaidzein.

Characterization of Alcaligenes faecalis strain AD15 indicating biocontrol activity against plant pathogens.

Bacterial strain possessing both bacteriostatic and fungistatic activity (biocontrol activity) against pathogens of cyclamen (Cyclamen sp.) was isolated from the soil in Gifu Prefecture, Japan, and characterized with respect to its taxonomic and biocontrol properties. The sequence of its 16S rRNA gene, morphology, biochemistry, and fatty acid composition demonstrated that it is a strain most closely related to Alcaligenes faecalis subsp. faecalis LMG 1229(T). The isolate was named A. faecalis strain AD15. A. faecalis AD15 produced hydroxylamine at maximum yields of 33.3±1.7 mg/L after 16 h cultivation in LB medium and 19.0±0.44 mg/L after 19 h cultivation in synthetic medium. Moreover, minimum inhibitory concentrations of hydroxylamine against the cyclamen pathogens Pantoea agglomerans and Colletotrichum gloeosporioides were 4.20±0.98 and 16.5±0.67 mg/L. These results indicated that the biocontrol activity of strain AD15 might be attributed to hydroxylamine, a metabolite in the culture medium, and it had the potential for biopesticide application.

Effect of the genistein metabolite on leptin secretion in murine adipocytes in vitro.

Soy isoflavonoids have many useful properties. However, they are metabolized in vivo, including in humans. The effect of the metabolism of soy isoflavonoids on their properties is not fully understood. We have isolated the bacterial strain SY8519, which has been shown to metabolize daidzein to O-desmethylangolensin and to produce 2-(4-hydroxyphenyl)propionic acid from genistein. According to chiral HPLC analysis, the 2-(4-hydroxyphenyl)propionic acid obtained from the bacterium was optically active. To determine the absolute stereochemistry of the microbial product, we prepared (S)-2-(4-hydroxyphenyl)propionic acid from (S)-2-phenylpropionic and concluded that the microbial product had an R-configuration by chiral HPLC analysis. We also applied the metabolite to mouse adipocytes and found that 2-HPPA was less effective at reducing leptin secretion than the parent compound genistein. Our results suggested that 'O-desmethylangolensin-production' attenuates the effect of soy isoflavonoids by reducing not only the activity of daidzein but also that of genistein.

Persimmon fruit tannin-rich fiber reduces cholesterol levels in humans.

Bile acid-binding agents are known to lower blood cholesterol levels and have been clinically used for the treatment of hypercholesterolemia. We previously showed that tannin-rich fiber from young persimmon (Diospyros kaki) fruits had bile acid-binding properties. In this study, we performed a randomized, double-blind, placebo-controlled trial to investigate the hypocholesterolemic effects of tannin-rich fiber in humans. The subjects (n = 40, plasma total cholesterol levels 180-259 mg/dl) were divided into 3 groups and ingested cookie bars containing 0 g (placebo group, n = 14), 3 g (low-dose group, n = 13), or 5 g (high-dose group, n = 13) of tannin-rich fiber 3 times daily before meals for 12 weeks. Plasma total cholesterol levels decreased significantly in the low-dose (12 weeks, p < 0.005) and high-dose (6 weeks, p < 0.05; 12 weeks, p < 0.001) groups. In addition, plasma low-density lipoprotein cholesterol levels decreased significantly in the high-dose group (6 weeks, p < 0.05; 12 weeks, p < 0.001). These improvements were not accompanied by changes in plasma high-density lipoprotein cholesterol or plasma triglyceride levels. Our findings indicate that tannin-rich fiber from young persimmon fruits is a useful food material for treating hypercholesterolemia.

Gating electrical transport through DNA molecules that bridge between silicon nanogaps.

DNA electronic devices were prepared on silicon-based three-terminal electrodes. Both ends of DNA molecules (400 bp long, mixed sequences) were bridged via chemical bonds between the source-drain nanogap (120 nm) electrodes. S-Shaped I-V curves were obtained and the electric current can be modulated by the gate voltage. The DNA molecules act as semiconducting p-type nanowires in the three-terminal device.

Induction of uncoupling protein-1 and -3 in brown adipose tissue by kaki-tannin in type 2 diabetic NSY/Hos mice.

Kaki-tannin, a highly polymerized-tannin from the young fruits of persimmon (Diospyros kaki 'Hachiya'), has been shown to have bile acid-binding activity. To verify the effect of kaki-tannin on the metabolism of lipid and glucose in type 2 diabetes, type 2 diabetic NSY/Hos mice were fed an AIN76-modified high fat diet supplemented with 1% (w/w) kaki-tannin for 8weeks. Kaki-tannin induced a 2-fold increase in fecal bile acid excretion and was significantly effective in the prevention of a rise in plasma cholesterol, triglyceride, and insulin levels. Kaki-tannin treatment also prevented fatty liver. To identify the molecular mechanism underlying these effects, gene expression analysis was performed on liver, brown adipose tissue (BAT), and skeletal muscle. The genes related to cholesterol metabolism, including 3-hydroxy-3-methylglutaryl-coenzyme A reductase and sterol regulatory element-binding protein 2, were increased in the liver of the kaki-tannin group. Interestingly, the uncoupling protein-1 (UCP1) gene and the UCP3 gene were significantly increased in the BAT of the kaki-tannin group, which was also confirmed at the protein level. These findings indicated that induction of UCP1 and UCP3 in the BAT by kaki-tannin treatment might influence the energy metabolism, thus contributing beneficial effects to type 2 diabetic NSY/Hos mice.

Complete genomic sequence of the O-desmethylangolensin-producing bacterium Clostridium rRNA cluster XIVa strain SY8519, isolated from adult human intestine.

The O-desmethylangolensin-producing Clostridium rRNA cluster XIVa strain SY8519 was isolated from the intestinal flora of a healthy human as a key isoflavonoid-metabolizing bacterium. Here, we report the finished and annotated genomic sequence of this organism.

Complete genomic sequence of the equol-producing bacterium Eggerthella sp. strain YY7918, isolated from adult human intestine.

Eggerthella sp. strain YY7918 was isolated from the intestinal flora of a healthy human. It metabolizes daidzein (a soybean isoflavonoid) and produces S-equol, which has stronger estrogenic activities than daidzein. Here, we report the finished and annotated genomic sequence of this organism.

Insulin receptor mutation results in insulin resistance and hyperinsulinemia but does not exacerbate Alzheimer's-like phenotypes in mice.

Obesity is a risk factor for Alzheimer's disease (AD), which is characterized by amyloid ß depositions and cognitive dysfunction. Although insulin resistance is one of the phenotypes of obesity, its deleterious effects on AD progression remain to be fully elucidated. We previously reported that the suppression of insulin signaling in a mouse with a heterozygous mutation (P1195L) in the gene for the insulin receptor showed insulin resistance and hyperinsulinemia but did not develop diabetes mellitus [15]. Here, we generated a novel AD mouse model carrying the same insulin receptor mutation and showed that the combination of insulin resistance and hyperinsulinemia did not accelerate plaque formation or memory abnormalities in these mice. Interestingly, the insulin receptor mutation reduced oxidative damage in the brains of the AD mice. These findings suggest that insulin resistance is not always involved in the pathogenesis of AD.

Coronary vein infusion of multipotent stromal cells from bone marrow preserves cardiac function in swine ischemic cardiomyopathy via enhanced neovascularization.

Few reports have examined the effects of adult bone marrow multipotent stromal cells (MSCs) on large animals, and no useful method has been established for MSC implantation. In this study, we investigate the effects of MSC infusion from the coronary vein in a swine model of chronic myocardial infarction (MI). MI was induced in domestic swine by placing beads in the left coronary artery. Bone marrow cells were aspirated and then cultured to isolate the MSCs. At 4 weeks after MI, MSCs labeled with dye (n=8) or vehicle (n=5) were infused retrogradely from the anterior interventricular vein without any complications. Left ventriculography (LVG) was performed just before and at 4 weeks after cell infusion. The ejection fraction (EF) assessed by LVG significantly decreased from baseline up to a follow-up at 4 weeks in the control group (P<0.05), whereas the cardiac function was preserved in the MSC group. The difference in the EF between baseline and follow-up was significantly greater in the MSC group than in the control group (P<0.05). The MSC administration significantly promoted neovascularization in the border areas compared with the controls (P<0.0005), though it had no affect on cardiac fibrosis. A few MSCs expressed von Willebrand factor in a differentiation assay, but none of them expressed troponin T. In quantitative gene expression analysis, basic fibroblast growth factor and vascular endothelial growth factor (VEGF) levels were significantly higher in the MSC-treated hearts than in the controls (P<0.05, respectively). Immunohistochemical staining revealed VEGF production in the engrafted MSCs. In vitro experiment demonstrated that MSCs significantly stimulated endothelial capillary network formation compared with the VEGF protein (P<0.0001). MSC infusion via the coronary vein prevented the progression of cardiac dysfunction in chronic MI. This favorable effect appeared to derive not from cell differentiation, but from enhanced neovascularization by angiogenic factors secreted from the MSCs.