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As it has been reported that type 2 diabetes mellitus increases the risk of Alzheimer’s disease, we investigated how to prevent type 2 diabetes and dementia using biofunctional boiled rice. We adopted unpolished super-hard rice (SHBR) for diabetes and wax-free unpolished black rice (WFBBR) for dementia and blended those with ordinary non-polished rice (KBR) (blending ratio 4:4:2), adding 2.5% waxy black rice bran (WBB) and 0.3% rice oil after high-pressure treatment (HPT) (WFBSK) to improve its palatability. This boiled rice is rich in dietary fiber, anthocyanin, free ferulic acid and β-secretase inhibitory activity. A randomized, parallel-group comparison study was conducted for 12 weeks with 24 subjects, using Cognitrax to evaluate their cognitive function primarily. Furthermore, as the secondary purpose, we performed a single-dose test for postprandial blood glucose and insulin secretion at the end of the human intervention test. After 12 weeks, consumers of the WFBSK rice exhibited significant improvement in language memory by cognitive test battery compared with those who consumed the control white rice (p < 0.05). Moreover, subjects who consumed the WFBSK rice had lower insulin secretion levels than those who consumed the control polished rice (p < 0.05).

Endothelial dysfunction is an early pathophysiological feature and independent predictor of a poor prognosis in most forms of cardiovascular disease. We evaluated the effect of brown rice crackers (BR-C) on endothelial function.Effect of heat-moisture treated (HMT) -BR-C on postprandial flow-mediated dilation (FMD) in adults with mild endothelial dysfunction was compared with that of BR-C and white rice crackers (WR-C) in 12 adults with mild endothelial dysfunction (less than 7.0% of FMD) by a randomized, single-blind, three-treatment three-period crossover trial (UMIN 000034898). Since we considered that the FMD increase was associated with the treatment of HMT-BR-C, we examined the effect of three possible factors: postprandial glucose levels, polyphenol content, and polyphenol release from the food matrix.Mean pre-intake baseline FMD values of HMT-BR-C, BR-C, and WR-C were 4.9%, 5.1%, and 4.9%, respectively, and those values 1 h post-intake were 6.3%, 5.1%, and 4.8%, respectively. There was no difference in intergroup comparisons of FMD using Dunnett's multiple comparison test. There was a significant increase in FMD only in HMT-BR-C in intragroup comparisons (HMT-BR-C intake increased the postprandial FMD response.

Reportedly, the intake of koji amazake, a beverage made from steamed rice fermented by Aspergillus oryzae, improves defecation frequency. However, its functional ingredients and mechanism of action remain unclear. To compare the effects of koji amazake and a placebo beverage on defecation frequency and to identify the functional ingredients and mechanism of action, a randomized, placebo-controlled, double-blind parallel-group comparative trial was performed on two groups. The koji amazake had 302 ± 15.5 mg/118 g of A. oryzae cells, which was not in the placebo. Compared with the placebo group, the koji amazake group showed a significant increase in weekly defecation frequency at 2 weeks (5.09 days vs. 4.14 days), 3 weeks (5.41 days vs. 4.18 days), and 4 weeks (5.09 days vs. 3.95 days), along with an increase in the weekly fecal weight at 4 weeks (724 g vs. 501 g). The intake of koji amazake did not induce significant intergroup differences in the fecal SCFA concentration, whereas it significantly decreased the relative abundance of Blautia and significantly increased that of Bacteroides at 3 weeks. Therefore, koji amazake intake improved defecation frequency, and A. oryzae cells played potentially important roles as functional ingredients.

Rice with double mutation of starch synthase IIIa and branching enzyme IIb (ss3a/be2b) has much higher amounts of apparent amylose and resistant starch (RS) than usual varieties. In this study, we conducted two randomized, single-blind, crossover trials to investigate the effect of single ingestion of two processed foods composed of ss3a/be2b mutant rice on postprandial blood glucose and insulin response in healthy adults, compared to those of usual cultivar. In trial 1, of ingestion of rice crackers, the incremental area under the curves of glucose (IAUCglc) and insulin (IAUCins) in RS group was significantly lower than in the control group. In trial 2, of the ingestion of cooked rice, IAUCglc in the RS group was significantly lower than in the control group. These results showed that the ss3a/be2b-mutant rice cracker and cooked rice having high RS can attenuate postprandial blood glucose and insulin response.

Introduction Oxygen has positive benefits and potentially damaging properties in biological systems. It is a highly reactive molecule, which participates in a high-energy electron transfer and through oxidative phosphorylation thereby supports generation of adenosine-5-triphosphate (ATP)

Background Metabolic syndrome is a major risk factor for a variety of obesity-related diseases. Recently, the effects of functional foods have been investigated on lipid metabolism as a means to reduce lipid content in the blood, liver and adipose tissues associated with carnitine O-palmitoyltransferase (CPT) activity. Acanthopanax senticosus (Rupr. et Maxim) Harms (AS) is a medicinal herb possessing a wide spectra of functions including antioxidant, anti-inflammatory and anti-fatigue actions. Despite much research being focused on the cortical roots of AS, little information is available regarding its leaves, which are also expected to promote human health, for example by improving abnormal lipid metabolism. Here, we explored whether AS leaves affect lipid metabolism in mice fed a high-fat diet. Results The administration of AS to BALB/c mice fed a high-fat diet significantly decreased plasma triglycerides (TG). CPT activity in the liver of these mice was significantly enhanced by AS treatment. Conclusion These findings indicate that AS leaves have the potential to alleviate increase in plasma TG levels due to high-fat diet intake in mice, possibly by increasing mitochondrial fatty acid β-oxidation, especially via CPT activation. Consequently, daily intake of AS leaves could promote beneficial health effects including the prevention of metabolic syndrome. © 2015 Society of Chemical Industry.

We analyzed high hydrostatic pressure (HP)-induced inactivation of Escherichia coli in the presence of sucrose, NaCl, KCl, or LiCl under 250–400 MPa. Salt stress response (osmotic or cationic) mechanism was demonstrated to be evaluated by HP-induced inactivation kinetics. We selected nine mutant strains from the Keio collection; five strains each lacked one osmoregulatory gene (ΔmzrA, ΔenvZ, ΔompR, ΔompC or ΔompF), and four strains each lacked one cationic stress-response gene (ΔmscS, ΔmscL, ΔchaA or ΔnhaA). The nine mutants exhibited three different patterns of HP-induced inactivation. Of the nine genes, four (mzrA, mscS, nhaA and mscL) were involved in piezotolerance at NaCl concentration of 0.0–0.35 M.

A high pressure (HP) tolerant (barotolerant) mutant a2568D8 and a variably barotolerant mutant a1210H12 were generated from Saccharomyces cerevisiae using ultra-violet mutagenesis. The two mutants, a barosensitive mutant a924E1 and the wild-type strain, were pressurized (225 MPa), and pressure inactivation behavior was analyzed. In the wild-type strain, a proportion of the growth-delayed cells were detected after exposure to HP. In a924E1, the proportion of growth-delayed cells significantly decreased compared with the wild-type. In a2568D8, the proportion of growth-delayed cells increased and the proportion of inactivated cells decreased compared with the wild-type. In a1210H12, the growth-delayed cells could not be detected within 120 s of exposure to HP. The proportion of growth-delayed cells, which incurred the damage, would affect the survival ratio by HP. These results suggested that cellular changes in barotolerance caused by mutations are remarkably affected by the ability to recover from cellular...

The cell cycle checkpoint system play a pivotal role in the cellular DNA damage response, and the discovery of checkpoint inhibitors is expected to sensitize current cancer therapies. Checkpoint signaling cascades are critically modulated by ATM (ataxia telangiectasia-mutated) and its related molecules. Generally, ATM primarily responds to ionizing irradiation-induced DNA double-strand breaks. Heavy ions from an accelerated carbon ion beam have been used to cure cancer because they are more effective than ionizing irradiation such as X-ray and γ-radiation in terms of biological damage. In a previous study, we demonstrated that a persimmon leaf flavonol (PLF) promoted the cytotoxic effect of chemotherapeutic agents on cancer cells through inhibition of checkpoint activities, especially in the ATM dependent pathway. The present study investigated whether PLF inhibits checkpoint activity during the DNA damage response induced by heavy ion irradiation. Treatment with PLF significantly increased the cytotoxicity of heavy ion irradiation in A549 adenocarcinoma cells. The phosphorylation of checkpoint proteins such as p53, SMC1, and Chk1 was increased by heavy ions. PLF reduced the phosphorylation of checkpoint proteins. Pre-treatment with PLF significantly prevented the decrease of mitotic cells in heavy ion-exposed cells. We further evaluated tumor volume in SCID mice inoculated with human lung adenocarcinoma A549 cells. The combination treatment of PLF and heavy ion resulted in a decrease of tumor volume compared with controls, although PLF itself did not exhibit any effect. These results indicate that PLF inhibits tumor growth through modulation of the DNA damage response. PLF may be useful for clinical application in combination with heavy ion radiotherapy.

The antihypertensive and vasorelaxant effects of water-soluble proanthocyanidins, extracted in persimmon leaf tea, were investigated in spontaneously hypertensive rats, rat aortas, and human umbilical vein endothelial cells. Oral administration of proanthocyanidins significantly decreased the systolic blood pressure of the rats after 4 h, as compared with distilled water controls. A vasorelaxant effect on rat aortas was induced by proanthocyanidins, and it was abolished by removal of the endothelium and inhibition of endothelial nitric oxide synthase and soluble guanylyl cyclase activity. The phosphorylation levels of endothelial nitric oxide synthase (Ser-1177) and the upstream kinase Akt (Ser-473) in umbilical cells also increased in a time-dependent manner after the addition of a proanthocyanidin-rich fraction. These results suggest that the antihypertensive effect of proanthocyanidins in persimmon leaf tea is due to vasorelaxation via an endothelium-dependent nitric oxide/cGMP pathway, and that proanthocyanidins might be useful in dietary lowering of blood pressure.

Persimmon leaf (PL) has been commonly recognized for its wide variety of health benefits. A previous study has reported that persimmon leaf extract (PLE) contained flavonols with the 2″-galloly moiety (PLEg). Galloylated homologues generically show stronger activity in their biological function, so enhanced functions can be expected for PLEg. We investigated in this present study the effect of PLEg on the cellular DNA damage checkpoint signaling to sensitize cancer chemotherapy. Treatment with PLE and PLEg significantly increased the cytotoxicity of doxorubicin (DOX) in A549 adenocarcinoma cells. PLE and PLEg reduced the phosphorylation of checkpoint proteins such as structural maintenance of chromosomes 1 (SMC1), checkpoint kinase 1 (Chk1), and p53 in DOX-treated cells. Moreover, PLE decreased the phosphorylation of ATM (ataxia telangiectasia mutated) in a dose-dependent manner. PLE, and especially PLEg, abrogated the G2/M checkpoint during DOX-induced DNA damage. These results suggest that PLEg specifically inhibited ATM-dependent checkpoint activation by DOX, and that PLEg might be a useful sensitizer in cancer chemotherapy.

Introduction Dried leaves of persimmon, traditionally consumed as a herbal tea in Japan, contain bioactive galloylated flavonol glycosides. Investigation of the seasonal compositional changes in these compounds is important for determining the optimum harvest time. Objective The three objectives were: (1) to elucidate the compositional changes of the flavonol glycosides in persimmon leaves collected in different seasons; (2) to develop an efficient method for separation of the constituents; and (3) to compare their antioxidant activities. Methodology Fresh persimmon leaves were collected at different growth stages in 2007 and 2008 in Niigata, Japan. Total flavonols were extracted with 70% aqueous acetone followed by ethyl acetate partitioning, and were analysed by high-performance liquid chromatography. Enzymatic transformation and chromatographic fractionation was performed to isolate the individual flavonols. Two antioxidant assays were performed. Results Four nongalloylated flavonol glycosides were detected at the leaf-shooting stage, and four additional galloylated flavonol glycosides accumulated during leaf development, resulting in a total of eight constituents. Isolation of nongalloylated and galloylated constituents was successfully achieved through enzymatic transformation of the flavonol mixture using combinations of tannase, β-glucosidase and β-galactosidase, followed by chromatographic fractionation. The gallates were identified as regiospecific 2″-galloylated galactosides and glucosides of kaempferol and quercetin. A mixture of 2″-galloylated flavonol glycosides had a two-fold stronger antioxidant activity than the nongalloylated mixture. Conclusion This study showed that four 2″-galloylated flavonol glycosides had accumulated in developing leaves of persimmon by the end of May through a rapid 2″-galloylation of the corresponding nongalloylated flavonol glycosides. Copyright © 2011 John Wiley & Sons, Ltd.

Using UV mutagenesis, a high pressure (HP)-sensitive (barosensitive) mutant of Saccharomyces cerevisiae was obtained. The mutant strain a924E1 showed a significant loss of viability at HP levels of 175 to 250 MPa at 20 °C compared with the parent strain. This strain also showed a significant loss of viability following heat treatment at 50–58 °C at 0.1 MPa. These results showed that the mutation caused a significant thermosensitivity as well as barosensitivity. The activation volume and activation energy values for the inactivation of strain a924E1 were equivalent to those of the parent strain. This suggested that the mechanism for the HP and thermal inactivation reaction of strain a924E1 was basically the same as that of the parent strain. Strain a924E1 showed no deficiency in growth and fermentation ability as well as auxotrophic property. Although the identification of the genetic sites of mutation introduced is underway, these phenotypes are favorable for the application of HP treatment and heat-assis...

Using UV mutagenesis, 2 high-pressure (HP) sensitive (barosensitive) mutants of Saccharomyces cerevisiae were obtained. The HP inactivation of the mutants, as well as their parent strains, followed 1st-order kinetics in the range of 175 to 250 MPa within 600 s. Both mutants showed larger 1st-order inactivation rate constant values or significant loss of viabilities, compared with their parent strains in the pressure range tested. The inactivation rate constant value of one of the mutants was comparable with that of a previously reported highly barosensitive strain, which was generated by deletion of hsp104 in a trehalose deficient strain. The activation volume values of HP inactivation reactions in the 2 mutants were apparently equivalent with those of their parent strains. This suggested that the mutation did not bring drastic volume changes of the key molecules for HP inactivation. Their auxotrophic properties, growth, and ethanol fermentation were identical in mutant and parent strains. The mutants could therefore be useful for fermentations where control by HP processing is desired.

The amounts and compositions of polyphenol in persimmon leaves and persimmon leaf tea were investigated. The predominant polyphenols in fresh leaves were water-soluble, and the contents reached a maximum (2.40% w/w) in June, and then gradually decreased. Separation of them followed by thiolytic degradation revealed that the major components were unique proanthocyanidin oligomers consisting of four heterogeneous extension units, including epigallocatechin-3-O-gallate. Persimmon leaf tea also contained similar proanthocyanidins with similar compositional units. Oral administration of starch with polyphenol concentrate of persimmon leaf tea resulted in a significant and dose-dependent decrease in the blood glucose level in Wistar rats. This effect is considered to be due to inhibition of pancreas alpha-amylase. These results indicate that persimmon leaf tea containing peculiar proanthocyanidins has a significant role in suppressing blood glucose elevation after starch intake, and that the best harvest time is June.

The purpose of this study was to examine the effects of gamma-aminobutyric acid (GABA) in fermented drinking water prepared from sodium glutamate, vinegar, and dried bonito (FDWG) compared with placebo [vinegar and dried bonito without GABA (FDW)] and its safety in normotensive and mildly or moderately hypertensive volunteers. A double-blind, placebo-controlled, randomized study was conducted involving volunteers with normal (group-N) and mildly or moderately high (group-H) blood pressure (BP). After a pretreatment period of 2 weeks (weeks -2), the subjects received FDWG or FDW for 12 weeks followed by 4 weeks of no intake (weeks 16). In group-H, both FDWG and FDW significantly decreased systolic (SBP, -7.6 +/- 4.0 and -5.5 +/- 1.5 mmHg, p0.05, respectively) and diastolic (DBP, -10.6 +/- 4.0 and -7.6 +/- 1.7 mmHg, p0.01, respectively) BP compared to the baseline (0-week) value at 12 weeks, respectively. There were no abnormal changes in hematological or blood chemistry variables, urinalysis, heart rate, or body weight in the study groups. These findings indicated that vinegar and dried bonito with or without GABA might have an effect on BP in mildly or moderately hypertensive patients.

The purpose of this study was to investigate body composition focusing on motor functional capacity and to correlate body composition with functional outcomes with subacute stroke patients. The subjects of this study were 34 inpatients with hemiparetic stroke who could walk. Motor functional capacity of patients was assessed on the basis of the Brunnstrom recovery stage (BRS), and functional status was measured using the Functional Independence Measure (FIM) instrument scores and gait velocity. Body composition was assessed by bioelectrical impedance analysis. Significant correlations were observed between FIM scores and the BRS of the lower extremity or lean body mass. The correlations for body water in affected and unaffected legs with the FIM subscores were statistically significant (p

Metabolic syndrome is a major risk factor for a variety of obesity-related diseases. Recently, the effects of functional foods have been investigated on lipid metabolism as a means to reduce lipid content in the blood, liver and adipose tissues associated with carnitine O-palmitoyltransferase (CPT) activity. Acanthopanax senticosus (Rupr. et Maxim) Harms (AS) is a medicinal herb possessing a wide spectra of functions including antioxidant, anti-inflammatory and anti-fatigue actions. Despite much research being focused on the cortical roots of AS, little information is available regarding its leaves, which are also expected to promote human health, for example by improving abnormal lipid metabolism. Here, we explored whether AS leaves affect lipid metabolism in mice fed a high-fat diet.The administration of AS to BALB/c mice fed a high-fat diet significantly decreased plasma triglycerides (TG). CPT activity in the liver of these mice was significantly enhanced by AS treatment.These findings indicate that AS leaves have the potential to alleviate increase in plasma TG levels due to high-fat diet intake in mice, possibly by increasing mitochondrial fatty acid β-oxidation, especially via CPT activation. Consequently, daily intake of AS leaves could promote beneficial health effects including the prevention of metabolic syndrome. © 2015 Society of Chemical Industry.

Many reports have described the bioavailability of anthocyanins; however, most of these reports investigated only the amount of anthocyanins excreted in urine. In the present study, we calculated the pharmacokinetic bioavailability of anthocyanins in rats by measuring the plasma concentration of delphinidin-3-rutinoside that had been administered orally or intravenously. Delphinidin-3-rutinoside was primarily absorbed in the blood and excreted into urine as unmetabolized forms with a T(max) of 26.3 min and a C(max) of 0.285 +/- 0.071 micromol/L. We detected small amounts of the metabolite 4'-O-methyl-delphinidin-3-rutinoside in the plasma, but we detected neither anthocyanidin (aglycone) nor glucuro- or sulfoconjugates. For the 8 h period after intake, delphinidin-3-rutinoside and 4'-O-methyl-delphinidin-3-rutinoside were excreted to urine at 795 +/- 375 and 12.3 +/- 2.91 nmol, respectively. Relative to intravenous injection, oral administration of delphinidin-3-rutinoside resulted in complete bioavailability (0.49 +/- 0.06%). Analysis of delphinidin-3-rutinoside plasma concentrations in bile cannulated rats revealed that, for the 8-h period after intake, the intact delphinidin-3-rutinoside excretion ratio in bile was 11% of the excretion ratio of 4'-O-methyl-delphinidin-3-rutinoside, 1.91 +/- 0.35 nmol versus 17.4 +/- 8.67 nmol, respectively. Setting the bile duct cannulation in a Bollman-type cage, however, significantly increased the bioavailability of orally administered delphinidin-3-rutinoside (18.14 +/- 6.24%). This effect appears to stem immobilization stress by reducing gastrointestinal motility. The cumulative excretion of delphinidin-3-rutinoside and 4'-O-methyl-delphinidin-3-rutinoside in urine and bile was 2.67 +/- 1.24% (w/w) of the dose ingested. Studies report that several metabolites are formed after oral ingestion of anthocyanins. Examples include glucuronyl from cyanidin-3-glucoside and both glucuronyl and sulfate conjugates from pelargonidin-3-glucoside. Our results indicate that delphinidin-3-rutinoside might be metabolized differently from cyanidin-3-glucoside and pelargonidin-3-glucoside.

For better understanding of the physiological function of anthocyanins, the absorption and metabolism of cyanidin 3-O-beta-D-glucopyranoside (Cy3G), which is one of the major anthocyanins in colored food materials, were precisely investigated. Combining two modalities newly developed, that is, highly sensitive semi-micro-HPLC and vein cannulation, Cy3G and its four major metabolites (M1-M4) were detected in the blood plasma of rats after oral administration of Cy3G (100 mg/kg of body mass). The plasma concentration of Cy3G reached its maximum at 15 min after the ingestion. Metabolite 2 (M2) and metabolite 3 (M3) showed their maximum plasma levels at 15 and 30 min, respectively, whereas metabolite 1 (M1) and metabolite 4 (M4) showed their maximum levels at 60 and 120 min, respectively. The maximum plasma concentrations of the four metabolites were in the following order: M3 (21 nM)M4 (20 nM)M1 (8.5 nM)M2 (5 nM). When Cy3G was directly injected into the neck vein, only M2 and M3 were detected in the plasma, indicating that both M1 and M4 were produced during absorption from the gastrointestinal tract. Tandem MS analysis of the metabolites showed that M2 and M3 were monomethylated Cy3G, while M1 and M4 were glucuronides of Cy and methylated Cy, respectively. M3 was assigned as peonidin 3-O-beta-D-glucopyranoside (Pn3G) from the comparison of the retention time of authentic Pn3G.

This paper reviews the evidence for novel physiological functions of oligosaccharides. A history of the research and development and the types of health benefits are discussed. A key property of the physiological functions lies in the indigestibility of oligosaccharides, which gives rise to fermentation in the large intestine, followed by an increase of Bifidobacteria and short-chain fatty acid (SCFA) production. This property leads to several kinds of physiological functions, classified into three types of health benefits. The first is the improvement of gastrointestinal conditions, including a normal stool frequency, less constipation, and healthy intestinal microflora. The second is the promotion of mineral absorption, including an increase of bone density and relief of anemia. The third is an immunomodulation effect, such as allergy and cancer prevention.

The purpose of this study was to investigate the relationship between daily physical activity and physical fitness in twenty ambulatory post-stroke patients. The pedometric measurement was made using a caloriecounter in daily life at home and assessed to quantify the time constant (τ on) of oxygen uptake (VO2) and oxygen deficit with gas analysis using a bicycle ergometer. There were negative correlations between the amount of daily walking steps and τ on, as well as the oxygen deficit at the onset of exercise (τ on; r=-0.52, p

Hemiparetic stroke patients have both decreased oxygen consumption and low endurance to exercise. This study examined their physical fitness to quantify the initial deficit and change in oxygen uptake kinetics (VO2 kinetics) during inpatients rehabilitation following stroke. Twenty-nine ambulatory hemiparetic stroke patients were treated in an 8-week program of low intensity aerobic exercise. They undertook a bicycle ergometer test with gas analysis before and after the exercise program to obtain maximum oxygen uptake (VO2 peak), maximum work load (max WR), and VO2 kinetics data. The time constant (τon) and O2 deficit on constant load after exercise training were significantly lower than those in initial measurement by 17% and 10%, respectively. On the incremental load, VO2 peak and max WR were significantly increased by 16% and 17% respectively and ΔVO 2/ΔWR was decreased by 11%. These results show that low-intensity endurance training produces energy efficiency of oxygen uptake kinetics and improves the physical fitness of ambulatory hemiparetic stroke patients.

The effects of acute or chronic intake of boysenberry juice or artificial vinegar on blood pressure (BP) and endothelial function were investigated in spontaneous hypertensive rats (SHR). A single administration of boysenberry juice (BJ, equivalent to 0.5 mL/kg body weight) or artificial boysenberry juice vinegar (BJV, equivalent to 0.5 mL BJ and 0.10 g acetic acid/kg body weight) decreased both systolic blood pressure (SBP) and diastolic blood pressure (DBP) significantly. Reductions in SBP of the control group compared with the BJ and BJV groups reached maxima of -16.8±4.3 and -28.4±7.3 mmHg 8 h after administration, respectively. Chronic SBP- and DBP-lowering effects were also observed upon daily feedings of both BJ and BJV at 4 wk. No significant differences were found in SBP or DBP between respective acute and chronic intake of BJ and BJV, except for the decrease in DBP after 4 wk of BJV intake. This suggests that the polyphenol constituents in BJ and BJV likely play a major role in lowering SBP and DBP under these conditions and that acetic acid added to BJ exerts a DBP-lowering effect after 4 wk of BJV intake. The polyphenolic constituents of these beverages might elevate plasma NO concentration via aortic endothelial nitric oxide synthase activation, but the effects of chronic intake on blood pressure might also be at least partly mediated by the renin-angiotensin system. These results may help explain the beneficial effects of boysenberry intake on cardiovascular health, such as reduced blood pressure and improved endothelial function.

Four anthocyanin components of black currant, delphinidin 3-O-beta-rutinoside (D3R), cyanidin 3-O-beta-rutinoside (C3R), delphinidin 3-O-beta-glucoside (D3G), and cyanidin 3-O-beta-glucoside (C3G), were successfully isolated as crystalline forms on a preparative scale. In this process, selective hydrolysis of the glucosides (D3G and C3G) and rhamnosides (D3R and C3R) was achieved by treatment with beta-glucosidase and hesperidinase (alpha-rhamnosidase), respectively, to improve resolution of anthocyanin components. Especially, selective conversion of the rutinosides into glucosides made the amounts of D3G and C3G increase about 4- and 7-fold, respectively. D3R, C3R, D3G, and C3G were isolated from enzymatic hydrolysates of black currant anthocyanins through Amberlite XAD-7HP absorption followed by preparative HPLC separation, and their crystals were obtained as the flavylium chloride.

Endoinulinase was partially purified from the culture filtrate of a filamentous fungus Aspergillus niger mutant 817. The enzyme preparation was immobilized covalently onto a porous cellulose derivative, Amino-Cellulofine. A 5% (w/v) solution (pH 5.0) of pure dahlia inulin was fed continuously into a packed-bed column reactor containing the immobilized enzyme. The operating conditions were studied to produce a mixture of oligomers with a degree of polymerization of 3 to 5 by partial hydrolysis of inulin. Inulotriose (F3) and -tetraose (F4) were purified from the hydrolysis products by preparative high-performance liquid chromatography. In vitro studies indicated that both the F3 and F4 were preferentially utilized by Bifidobacterium spp., but not by Escherichia coli or Clostridium perfringens.

The fopA gene encoding a fructooligosaccharide-producing beta-fructofuranosidase was isolated from Aspergillus niger ATCC 20611. The primary structure deduced from the nucleotide sequence showed considerable similarity to those of two other beta-fructofuranosidases from A. niger, but the fopA gene product had several amino acid insertions and an extra C-terminal polypeptide consisting of 38 amino acids that could not be found in the two others. We could successfully express the fopA gene in S. cerevisiae and the fopA gene product obtained from the culture supernatant of the S. cerevisiae transformant had similar characteristics to the beta-fructofuranosidase purified from A. niger ATCC 20611. However, we could not detect any beta-fructofuranosidase activity in either the culture supernatant or cell lysate when the C-terminal truncated fopA gene product by 38 amino acids was used to transform S. cerevisiae. In western analysis of those samples, there was no protein product that is cross-reacted with anti-beta-fructofuranosidase antibody. These results suggested that the C-terminal region of the fopA gene product consisting of 38 amino acids was essential for the enzyme production.

Previously, we confirmed that dietary fructooligosaccharides (FOS) increase calcium absorption in rats. In this study, we examined the influence of FOS feeding on the concentration of calbindin-D9k of several intestinal segments in rats. Rats in the control group were fed a diet without FOS. Rats in the other two groups were fed the diet containing FOS at either 50 or 100 g/kg for 10 d and subjected to a calcium absorption study. On the final day of feeding, the rats were killed and the entire intestine was removed. The intestinal mucosa was collected from four segments, i.e., the proximal and distal segments of the small intestine, the cecum and the colorectum, respectively. The apparent absorption of calcium increased dose dependently (r = 0.9256, P < 0.0001). Significant positive correlations between apparent calcium absorption and the relative amounts of calbindin in both large intestinal segments were observed (cecum, r = 0.8956, P = 0.0011; colorectum, r = 0.8828, P = 0.0016). Also, significant negative correlations between apparent calcium absorption and the relative amounts of calbindin-D9k in both small intestinal segments were observed (proximal, r = -0.7149, P = 0. 0304; distal, r = -0.8740, P = 0.0021). In conclusion, FOS feeding increases levels of calbindin-D9k in the large intestine, but decreases those in the small intestine. Moreover, these results suggest that part of the stimulatory effect of fructooligosaccharides relates to the transcellular route of calcium absorption in the large intestine of rats.

Gastrectomized rats develop anemia and osteopenia, and ingestion of fructooligosaccharides leads to an increase in iron absorption and promotes recovery from anemia in iron-deficient rats. Laparotomized (sham-operated control) rats and totally gastrectomized (Billoth II) rats, in groups of 14 each, were fed a control diet without fructooligosaccharides or a diet containing fructooligosaccharides (75 g/kg of diet) for 6 wk. All rats received an intramuscular injection of vitamin B-12 every 2 wk. Tail blood was collected every week for determination of hematocrit and hemoglobin concentration. At the end of the experiment, the rats were killed and the femur and tibia were collected for measurement of bone mineral density (BMD). The hematocrit, hemoglobin concentration, hemoglobin regeneration efficiency, and BMD of both femurs and tibias were significantly lower in gastrectomized rats fed the control diet than in the other three groups. Dietary fructooligosaccharides prevented anemia and osteopenia in totally gastrectomized rats.

The influence of fructo-oligosaccharides of different sugar chain length on growth, nitrogen balance, and mineral balance was examined in rats fed diets containing either sucrose, 1-kestose, nystose, or a mixture of these at 50 or 100 g/kg of diet. Rats were fed the experimental diets for six weeks, and nitrogen balance and mineral (Ca and Mg) balance were examined after the second and fourth week. Food intake and body weight gain did not differ significantly among the groups. Each fructo-oligosaccharide fed was found to increase Ca and Mg absorption dose-dependently during the first balance study period and the degrees of increase in Ca and Mg absorption were similar with each fructo-oligosaccharide fed regardless of the level present in the experimental diet. Also, each of the fructo-oligosaccharides was found to increase fecal excretion of nitrogen, but there was no clear dose-dependence and the extent of increase in fecal excretion of nitrogen was similar for each fructo-oligosaccharide fed. In conclusion, our findings demonstrate that ingestion of fructo-oligosaccharides leads to increases in Ca and Mg absorption and an increase in fecal excretion of nitrogen, however, differences in sugar chain length do not influence their nutritional effects considerably in rats.

There is an increasing interest in dietary polyphenols for risk reduction in cardiovascular diseases. The aim of this study was to evaluate acute and chronic flow-mediated dilation (FMD) and blood pressure responses to daily intake of boysenberry juice. FMD of the brachial artery was measured in six subjects in the initial, intermediate and follow-up stages of a 4-week open-label intervention study. The intake of boysenberry juice (180 ml/d) increased FMD with progression of intervention stage, and FMD differed in the follow-up stage compared with pre-intake baseline (p = 0.0163

カゼインホスホペプチド(CPPs:α-CPP及びβ-CPPの混合物)が,ウシ精子のウシ体外成熟卵子への侵入(体外受精)およびその後の胚発生に及ぼす影響について検討した.受精試験には,通常の体外受精方法では受精率が低いウシAおよびBならびに受精率が高いウシCの凍結融解精子を用い,体外成熟卵子への精子侵入率(受精率)を調べた.体外受精用培養液には,カフェイン(5mM),ヘパリン(10単位/ml)を添加し,実験区にはさらにCPPs(1mg/ml)を添加した.その結果,体外受精培養液にCPPsを添加することによって,ウシAおよびBにおいて有意に高い受精率が得られた:ウシA(添加79%,無添加38%),ウシB(添加45%,無添加13%)(P

A plasmid pLC-bar containing the bialaphos resistance gene derived from Streptomyces hygroscopicus between the Lentinus edodes ras gene promoter and priA gene terminator was constructed. When protoplasts of Pleurotus ostreatus were mixed with the plasmid DNA in the presence of polyethylene glycol and CaCl2, bialaphos-resistant colonies were obtained. This indicated that transformation was successful. Southern blot analysis of total DNAs from transformants showed that the introduced plasmid DNA was integrated into the host chromosome and partly rearranged. A plasmid, pLC-GUS, containing the Escherichia coli beta-glucuronidase (GUS) gene under the control of the L. edodes ras gene promoter and priA gene terminator was constructed and introduced into protoplasts of P. ostreatus with pLC-bar by co-transformation. Two of 5 transformants obtained as bialaphos-resistant colonies showed two to twenty times higher specific activity of GUS than the recipient. Southern blot analysis of total DNAs from transformants indicated the presence of the GUS gene only in the two transformants. These results indicated that co-transformation of P. ostreatus was successful, and that the GUS gene was expressed in P. ostreatus. This transformation system will enable us to breed commercial strains of P. ostreatus at the molecular level.

The antihypertensive effect of a single oral administration of a boysenberry seed polyphenol extract to spontaneously hypertensive rats was evaluated at different doses (100 and 200 mg/kg), and a significant decrease in systolic blood pressure (SBP) was observed up to 6 h post administration. The extract was separated into proanthocyanidin-rich and ellagitannin fractions by solvent partition. A significant decrease in SBP was observed only after administering the proanthocyanidin-rich fraction, and this decrease was abolished by an N(G)-nitro-L-arginine methyl ester (L-NAME) injection. An analysis of the orally absorbable components showed that intact dimeric and trimeric procyanidins and propelargonidins were detectable in the plasma with a maximal concentration 2 h post administration. The vasorelaxant activity of the extract was also confirmed by in vitro assay using rat aorta rings. These results suggest that proanthocyanidins (PAs) in boysenberry seeds may have played an important role in the observed antihypertensive effect.

Using microplates as pressure and cultivation vessels, a high-throughput method was developed for analyzing the high-pressure inactivation kinetics of microorganisms. The loss of viability from a high-pressure treatment, measured based on the growth delay during microplate cultivation, showed reproducibility with the conventional agar plate method and was applicable for the kinetics analysis.

Mouse embryonic stem cells (ESCs) can be induced to form pancreatic exocrine enzyme-producing cells in vitro in a stepwise fashion that recapitulates the development in vivo. However, there is no protocol for the differentiation of pancreatic-like cells from human ESCs (hESCs). Based upon the mouse ESC model, we have induced the in vitro formation of pancreatic exocrine enzyme-producing cells from hESCs. The protocol took place in four stages. In Stage 1, embryoid bodies (EBs) were formed from dissociated hESCs and then treated with the growth factor activin A, which promoted the expression of Foxa2 and Sox17 mRNAs, markers of definitive endoderm. In Stage 2, the cells were treated with all-trans retinoic acid which promoted the transition to cells that expressed gut tube endoderm mRNA marker HNF1b. In Stage 3, the cells were treated with fibroblast growth factor 7 (FGF7), which induced expression of Pdx1 typical of pancreatic progenitor cells. In Stage 4, treatment with FGF7, glucagon-like peptide 1, and nicotinamide induced the expression amylase (AMY) mRNA, a marker for mature pancreatic exocrine cells. Immunohistochemical staining showed the expression of AMY protein at the edges of cell clusters. These cells also expressed other exocrine secretory proteins including elastase, carboxypeptidase A, chymotrypsin, and pancreatic lipase in culture. Production of these hESC-derived pancreatic enzyme-producing cells represents a critical step in the study of pancreatic organogenesis and in the development of a renewable source of human pancreatic-like exocrine cells.

Proanthocyanidins and other polyphenols in the seeds and juice of boysenberry were quantitatively analyzed. Polyphenolic extracts were prepared from the waste seeds and commercial juice by chromatographic fractionation. Compositional analysis revealed that both extracts contained six polyphenolic classes: flavanol monomers, proanthocyanidins, anthocyanins, ellagic acid, ellagitannins, and flavonol glycosides. Ellagitannins were the most abundant polyphenols in both extracts. Proanthocyanidins were present as short oligomers consisting of dimeric and trimeric procyanidins and propelargonidins, with the most abundant component being procyanidin B4 in both extracts. Quantification by high-performance liquid chromatography-mass spectrometry (HPLC-MS) revealed that the seeds contained a 72-fold higher amount of proanthocyanidins than the juice. These results indicate that boysenberry fruits contain short oligomeric proanthocyanidins along with flavanol monomers and the seeds represent a good source of short oligomeric proanthocyanidins.