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The PtO2-catalyzed hydrogenation of curcumin produced slightly predominant meso-octahydrocurcumin than raceme octahydrocurcumin. Similar result was found in the product obtained from tetrahydrocurcumin and NaBH4, whereas using palladium carbon as a catalyst increased the meso-octahydrocurcumin ratio. Compared with chemical methods, baker's yeast produced 3S,5S-octahydrocurcumin and meso-octahydrocurcumin from tetrahydrocurcumin. The different activity between raceme and meso-octahydrocurcumin was not found in our experiments.

Supplementary Data from A Link between Benzyl Isothiocyanate-Induced Cell Cycle Arrest and Apoptosis: Involvement of Mitogen-Activated Protein Kinases in the Bcl-2 Phosphorylation

In the present study, we clarified the molecular mechanism underlying the relationship between benzyl isothiocyanate (BITC)-induced cell cycle arrest and apoptosis and the involvement of mitogen-activated protein kinases (MAPKs). The exposure of Jurkat human T-cell leukemia cells to BITC resulted in the inhibition of the G2-M progression that coincided with the apoptosis induction. The experiment using the phase-specific synchronized cells demonstrated that the G2-M phase-arrested cells are more sensitive to undergoing apoptotic stimulation by BITC than the cells in other phases. We also confirmed that BITC activated c-Jun N-terminal kinase (JNK) and p38 MAPK, but not extracellular signal-regulated kinase, at the concentration required for apoptosis induction. An experiment using a JNK-specific inhibitor SP600125 or a p38 MAPK inhibitor SB202190 indicated that BITC-induced apoptosis might be regulated by the activation of these two kinases. Conversely, BITC is likely to confine the Jurkat cells in the G2-M phase mainly through the p38 MAPK pathway because only the p38 MAPK inhibitor significantly attenuated the accumulation of inactive phosphorylated Cdc2 protein and the G2-M-arrested cell numbers. We reported here for the first time that the antiapoptotic Bcl-2 protein was phosphorylated by the BITC treatment without significant alteration of the Bcl-2 total protein amount. This was abrogated by a JNK specific inhibitor SP600125 at the concentration required for specific inhibition of the c-Jun phosphorylation. Moreover, the spontaneous phosphorylation of antiapoptotic Bcl-2 in the G2-M synchronized cells was enhanced synergistically by the BITC treatment. Involvement of the MAPK activation in the Bcl-2 phosphorylation and apoptosis induction also was observed in HL-60 and HeLa cells. Thus, we identified the phosphorylated Bcl-2 as a key molecule linking the p38 MAPK-dependent cell cycle arrest with the JNK activation by BITC.

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.

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, 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

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.

The previous slide-glass type system could simultaneously detect reactive and highly reactive oxygen species, i.e., superoxide radicals (O2−·) and hypochlorite ions (OCl−) elicited from leucocytes in sample blood, but had some drawbacks, i.e., signal noise from air-flow stirring, potential biohazard risks, etc. because of open samples placed on a slide glass. We overcame these drawbacks by adopting a fluidic-chip container in a new system, which resulted in higher sensitivity and more stable measurements. Using the new system, we conducted a pilot study on nominally healthy volunteers to find whether or not the monitored activities of leukocytes can distinguish more or less unhealthy conditions from healthy ones. At first, healthy volunteers of both genders and of various ages showed that the fluctuation magnitudes (%) of O2−· and OCl− were nearly similar to each other and to that of the neutrophil count fluctuation. These parameters sometimes exceeded the healthy fluctuation range. By comparing these large fluctuations with the data of an inflammation marker C-reactive protein (CRP), the neutrophil count fluctuation and the timings/symptoms of abnormalities found in questionnaire, we could gain information suggesting the factors causing the large fluctuations. The new system could detect bodily abnormalities earlier than CRP or self-aware symptoms.

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.

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 ATCC14025

Lipid peroxidation is known to be a free chain reaction which takes place both in vivo and in vitro, and forms lipid hydroperoxides and secondary products. These lipid peroxidation products are highly reactive and have been shown to interact with many biological components such as proteins, amino acids, amines, phospholipids and DNAs. As novel biomarkers for early stage of lipid peroxidation process, we have developed monoclonal antibodies specific to oxidatively modified lysines with linoleic acid-, arachidonic acid- eicosapentanoic acid- and docosahexaenoic acid-hydroperoxides. We have also succeeded in construction of novel biomarkers specific to overproduction of oxidative stress during inflammation process. Recently, we found more potent antioxidative phytochemicals such as sesame lignans, astaxanthin and curcuminoids, especially, tetrahydrocurcumin , one of the main metabolites and also produced by fermentation of yeast (D. hansenil) of curcumin. We have also succeeded in determining many functional activities including extention of ageing process.

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 showed25% 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.

Oxidative stress may cause a wide variety of free radical reactions to produce deleterious modifications in membranes, proteins, enzymes, and DNA. Reactive Oxygen Species (ROS) generated by myeloperoxidase (MPO) can induce lipid peroxidation and also play an important role in the generation of reactive chlorinating and brominating species. As the universal biomarkers, chemical, and immunochemical approach on oxidatively modified and halogenated tyrosines has been carried out. As amido-type adduct biomarkers, chemical, and immunochemical evaluation of hexanoyl- and propanoyl-lysines, hexanoyl- and propanoyl-dopamines and phospholipids were prepared and developed for application of evaluation of novel antioxidative functional food factors. We have also involved in application of oxidatively modified DNAs such as 8-hydroxy- and 8-halogenated deoxyguanosines as the useful biomarkers for age-related diseases using both in vitro and in vivo systems. Application of these oxidative stress biomarkers for novel type of functional food development and recent approach for development of novel evaluation systems are also discussed.

Oxidative stress has been implicated in various disorders and controlling it would be important for healthy life. We have developed a new optical system for easily and accurately measuring oxidative stress in whole blood. It is optimized for simultaneously detecting reactive oxygen species (ROS) and highly reactive ROS (hROS), elicited mostly by white blood cells in a few microliters of blood. Results obtained by using this system show at least four important findings. 1) chemiluminescence of MCLA was confirmed to be attributable to O2-•. 2) PMA-stimulated cells released O2-• longer and more slowly than fMLP-stimulated ones. 3) fluorescence produced by APF oxidation was confirmed to be attributable to hROS, mostly OCl-, produced by myeloperoxidase. 4) the generation of OCl- was found to be a slower process than the O2-• generation. We also conducted pilot studies of oxidative stress in healthy volunteers.

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.

We tried to isolate metabolite in curcumin by incubation with human feces in vitro for the better understanding of curcumin activity in vivo. One group containing some bacteria, referred as 1C, produced a metabolite other than tetrahydrocurcumin. Structural analysis revealed that this new metabolite was 3-hydroxy-1,7-bis(3,4-dihydroxyphenyl)heptane by comparing it to the reported optically active compound rubranol, which has 3R stereochemistry. In order to determine the absolute stereochemistry of the secondary alcohol of this metabolite, we synthesized its racemic tetramethyl derivative. From HPLC analysis that separate the synthetic sample to two peaks, this metabolite was optically active and was primarily an enantiomer of rubranol. We also compared the activities of curcumin and the metabolite using monocyte chemoattractant protein-1 (MCP-1) which is produced in vitro by murine adipocytes. The metabolite exhibited an inhibition effect associated with the production of MCP-1, which was similar to that of curcumin with attenuated cytotoxicity.

Functional food ingredients, including prebiotics, have been increasingly developed for human health. The improvement of the human intestinal environment is one of their main targets. Fructooligosaccarides (FOS) are oligosaccharide fructans that are well studied and commercialized prebiotics. 1-Kestose, one of the components of FOS, is considered to be a key prebiotic component in FOS. However, to our knowledge, no studies have been reported on the physiological efficacy of 1-Kestose regarding its anti-oxidative activity. In the present study, we examined the effects of dietary 1-Kestose on gene expression of antioxidative enzymes in the liver, kidney and epididymal adipose tissue of mice by quantitative RT-PCR (qRT-PCR). We demonstrated that a 1-Kestose-rich diet increased mRNA and enzymatic activity levels of glutathione-S-transferase (GST) in mouse liver. These results suggest the possibility that dietary 1-Kestose as a prebiotic may enhance antioxidative activity in mice.

Maturation and aging induce alterations in glucose and protein metabolism, which are responsible for insulin resistance and sarcopenia. In the present study, we examined the effects of long-term (16 weeks) ingestion of diets supplemented with tetrahydrocurcumin (THC) and/or branched-chain amino acids (BCAAs) on glucose tolerance and soleus muscle protein content in mature rats (28 weeks of age). Intraperitoneal glucose tolerance tests (IPGTTs) were performed at week 6 and week 12 during the experimental period. Glucose tolerance was not affected by 6-week supplementation with THC and/or BCAAs, but was improved by supplementation at 12 weeks. A synergistic effect of THC and BCAAs was not observed. The protein content of the soleus muscle was increased by long-term supplementation with BCAAs, but not THC. These results suggest that THC and BCAAs are potentially beneficial supplements to improving maturation (aging)-related metabolic deterioration.

Two potent antioxidative isoflavones were isolated from soybeans fermented with Aspergillus saitoi by silica gel column chromatography and preparative HPLC, using ODS column, or in addition, Toyopearl HW-40 column chromatography. The purified AS-13 and AS-9B compounds were identified as 8-hydroxydaidzein (8-OHD) and 8-hydroxygenistein (8-OHG), respectively, by MS, and (1)H-NMR, (13)C-NMR and HMBC spectra. These isoflavones, which have an o-dihydroxy structure between the 7- and 8-position, each exhibited significantly stronger antioxidative activity than daidzein and genistein in both oil and lipid/aqueous systems. Furthermore, the antioxidative activity and the content of each isoflavone analog in soybeans with different fermentation periods were investigated. It is suggested from these results that AS-13 and AS-9B were produced from daidzein and genistein, respectively, by hydroxylation at the 8-position of each isoflavone structure. In addition, it is concluded that these isoflavones were also the principal antioxidants in potent antioxidative soybeans fermented with A. saitoi.

The formation mechanism for the potent antioxidative o-dihydroxyisoflavones, 8-hydroxydaidzein (8-OHD) and 8-hydroxygenistein (8-OHG), was studied by incubating whole soybeans in a solid culture and a soybean extract in a liquid culture with Aspergillus saitoi. Analyses of changes in the isoflavone analogue content, β-glucosidase activity, and isoflavone hydroxylation ability indicated that 8-OHD and 8-OHG were formed from daidzein and genistein, respectively, by microbial hydroxylation, being respectively liberated from daidzin and genistin by β-glucosidase from A. saitoi during incubation. No selective hydroxylation reaction at the 8-position of daidzein and genistein were apparent during the vegetative stage, but were induced at the stage of sporulation.

Epidemiological studies present the beneficial effects of dietary habits on prevention of aging-associated decline of brain function. Phytochemicals, the second metabolites of food, protect neuronal cells from cell death in cellular models of neurodegenerative disorders, and the neuroprotective activity has been ascribed to the anti-oxidant and anti-inflammatory functions. In this paper, the cellular mechanism of neuroprotection by phytochemicals was investigated, using the cellular model of mitochondrial apoptosis induced by PK11195, a ligand of outer membrane translocator protein, in SH-SY5Y cells. PK11195 induced mitochondrial membrane permeabilization with rapid transit production of superoxide (superoxide flashes) and calcium release from mitochondria, and activated apoptosis signal pathway. Study on the structure–activity relationship of astaxanthin, ferulic acid derivatives, and sesame lignans revealed that these phytochemicals inhibited mitochondrial membrane permeabilization and protected cells from apoptosis. Ferulic acid derivatives and sesame lignans inhibited or enhanced the mitochondrial pore formation and cell death by PK11195 according to their amphiphilic properties, not directly depending on the antioxidant activity. Regulation of pore formation at mitochondrial membrane is discussed as a novel mechanism behind neuroprotective activity of phytochemicals in aging and age-associated neurodegenerative disorders, and also behind dual functions of phytochemicals in neuronal and cancer cells.

Rasagiline, a neuroprotective inhibitor of type B monoamine oxidase, prevented PK111195-induced apoptosis in SH-SY5Y cells through inhibition of mitochondrial apoptosis signaling (J Neural Transm 120:1539–1551, 2013, J Neural Transm 122:1399–1407, 2015). This paper presents that PK11195 induced superoxide flashes, the transit production burst, mediated by cyclosporine A-sensitive membrane permeability transition. Rasagiline prevented superoxide flashes, calcium efflux, and cell death by PK11195. Regulation of the initial pore formation at the inner mitochondrial membrane was confirmed as the decisive mechanism of neuroprotection by rasagiline.

Summary Background. Photoageing of skin is thought to be caused by protein denaturation, which can be induced by ultraviolet radiation. Previous studies have also reported that inflammation is related to protein denaturation; however, the influence of inflammation on skin ageing has not been explored in detail. Aim. To investigate the possible connection between inflammation and protein denaturation, which might lead to skin ageing, we focused on halogenated tyrosine as a denatured substance produced during the inflammation process. Methods. We measured halogenated tyrosine in aged human skin. Inflammatory cells and halogenated tyrosine were detected by immunohistochemistry using antibodies to mast-cell tryptase, neutrophilic myeloperoxidase and halogenated tyrosine. Finally, using elastic van Gieson (EVG) staining, we investigated whether the sites of halogenated tyrosine coincided with the sites at which proteins were denatured. Results. Immunohistochemical analysis indicated that both inflammatory cells and halogenated tyrosines increased with ageing in both photoexposed and photoprotected skin. EVG staining confirmed that the localization of halogenated tyrosine was close to the sites at which protein was denatured. Conclusions. Our investigations indicate a possible connection between skin ageing and inflammation, suggesting that halogenated tyrosine could be a useful marker of ageing skin.

The O-type forkhead domain transcription factor (FOXO) is involved in many biological processes such as aging, the oxidative stress response, and growth regulation. FOXO activity is tightly controlled within cells. In particular, growth factor signaling pathways and the oxidative stress response can both stimulate nuclear translocation of this transcription factor. Here, we show that tetrahydrocurcumin (THC), a curcumin metabolite, regulates the oxidative stress response and aging via FOXO. In NIH3T3 cells, THC induced nuclear accumulation of FOXO4, a member of the FOXO family of transcription factors, by inhibiting phosphorylation of protein kinase B (PKB)/Akt. In Drosophila melanogaster, THC attenuated the oxidative stress response, an effect that was blocked in a foxo mutant background. THC also extended the life span of Drosophila under normal conditions, and loss of either foxo or Sir2 activity eliminated this effect. Based on these results, THC may regulate the aging process via an evolutionarily conserved signaling pathway that includes both foxo and Sir2.

Daidzein and genistein are the main aglycones of soy isoflavonoid, and have many useful activities in vitro and in vivo . However, equol, a metabolite of daidzein in vivo , has attracted attention due to its stronger activity than that of the naturally occurring isoflavonoids. We subjected the soy isoflavonoids, including the naturally occurring ( S )-equol, to mouse adipocytes, and compared the inhibitory activity on the leptin secretion. Equol, daidzein and genistein inhibited the leptin secretion, whereas O -desmethylangolensin had a lower activity. The inhibitory activity of the isoflavones was not affected by the addition of an iNOS inhibitor and an estrogen.

The expression of cell adhesion molecules (CAMs) has been implicated as one of the most important causes of the development of inflammatory diseases such as atherosclerosis, and, it is speculated that the prevention of it is an effective approach to the control of atherosclerosis. In the present study, we investigated the effect of sesame lignans on the expression of CAMs in human umbilical vein endothelial cells (HUVECs) induced by tumor necrosis factor-alpha (TNF-alpha). Based on cell-ELISA analysis, we found that sesaminol-6-catechol downregulated the TNF-alpha-induced expression of CAMs in a dose-dependent manner. Moreover, these inhibitory effects were caused to be drastically exerted by downregulating the CAM proteins in TNF-alpha-activated HUVECs at transcriptional level. This suggests, that sesaminol-6-catehcol suppresses the expression of CAMs, and may be an active component of sesame lignans.

In a previous study we prepared monoclonal antibody against allyl isothiocyanate (AITC)-modified lysine (Lys), and found that AITC reacted with Lys under physiological conditions in vitro (T. Nakamura et al., Chem. Res. Toxicol., 22, 536-542 (2009)). In the present study, antibodies against benzyl isothiocyanate (ITC), 6-methylsulfinylhexyl ITC and phenethyl ITC modified protein were prepared, and the respective monoclonal antibodies, B6C9, 6MS3D10, and PE3A10 were obtained. These antibodies were applied to ITC detection in food using shredded Wasabia japonica (wasabi) and ground Carica papaya (papaya) seed by trapping ITC with biotin-labeled bovine serum albumin. ITC formation from the wasabi and papaya seed samples was confirmed using the antibodies in a dose-dependent manner. These antibodies might be applicable in identifying food-derived ITC.