Toshiki Enomoto

Isolation of a New Phlorotannin, a Potent Inhibitor of Carbohydrate-Hydrolyzing Enzymes, from the Brown Alga Sargassum patens

Ethanol extracts from 15 kinds of marine algae collected from the coast of the Noto Peninsula in Japan were examined for their inhibitory effects on human salivary α-amylase. Four extracts significantly suppressed the enzyme activity. An inhibitor was purified from the extract of Sargassum patens . The compound was a new phloroglucinol derivative, 2-(4-(3,5-dihydroxyphenoxy)-3,5-dihydroxyphenoxy) benzene-1,3,5-triol (DDBT), which strongly suppressed the hydrolysis of amylopectin by human salivary and pancreatic α-amylases. The 50% inhibitory activity (IC(50)) for α-amylase inhibition of DDBT (3.2 μg/mL) was much lower than that of commercially available α-amylase inhibitors, acarbose (26.3 μg/mL), quercetagetin (764 μg/mL), and α-amylase inhibitor from Triticum aestivum (88.3 μg/mL). A kinetic study indicated that DDBT was a competitive α-amylase inhibitor with a K(i) of 1.8 μg/mL. DDBT also inhibited rat intestinal α-glucosidase with an IC(50) value of 25.4 μg/mL for sucrase activity and 114 μg/mL for maltase activity. These results suggest that DDBT, a potent inhibitor of carbohydrate-hydrolyzing enzymes, may be useful as a natural nutraceutical to prevent diabetes.

Characterization of vitamin B12 compounds from Korean purple laver (Porphyra sp.) products.

Vitamin B(12) contents of various Korean purple laver products were determined with the microbiological vitamin B(12) assay method. Although a substantial amount (133.8 microg/100 g) of vitamin B(12) was found in dried purple laver, seasoned and toasted laver products contained lesser vitamin B(12) contents (about 51.7 microg/100 g). The decreased vitamin B(12) contents in the seasoned and toasted laver products, however, were not due to loss or destruction of vitamin B(12) during the toasting process. Silica gel 60 thin layer chromatography-bioautogram analysis indicated that all Korean laver products tested contain true vitamin B(12), but not inactive corrinoid compounds. In vitro gastrointestinal digestion experiments indicated that digestion rate of vitamin B(12) from the dried Korean purple laver was estimated to be 50% under pH 2.0 conditions (as a model of normal gastric function). These results suggest that Korean purple laver products would be excellent vitamin B(12) sources for humans, especially vegetarians.

Characterization of vitamin B 12 compounds from edible shellfish, clam, oyster, and mussel

Vitamin B 12 content of various edible shellfish was determined by both Lactobacillus leichmannii ATCC 7830 microbiological and intrinsic factor-chemiluminescence methods. The values determined by the microbiological method were 1.2-19.8 (M/C ratio) fold greater in the shellfish than the values determined by the chemiluminescence method. Vitamin B 12 compounds were purified from most eaten shellfish, oyster (M/C, 1.5), mussel (M/C, 1.2), and short-necked clam (M/C, 2.7), and partially characterized. TLC and HPLC patterns of each red-colored vitamin B 12 compound (M/C, 1.0-1.2) purified from these shellfish were identical to those of authentic vitamin B 12. Although the higher values in the determination of vitamin B 12 by the microbiological method may be due to the occurrence of vitamin B 12 -substitutive compounds, the edible shellfish would be excellent vitamin B 12 sources judging from the values (≧ 6 μ g/100g) determined by the chemiluminescence method.

Pyrosequencing Analysis of Microbiota in Kaburazushi, a Traditional Medieval Sushi in Japan

The processing of archetypal Japanese sushi involves microbial fermentation. The traditional sushi kaburazushi, introduced in the middle ages, is made by fermenting salted yellow tail, salted turnip, and malted rice, and is distinguished from the ancient sushi narezushi, made from fish and boiled rice. In this study, we examined changes in the microbial population during kaburazushi fermentation by pyrosequencing the 16S ribosomal RNA genes (rDNA) of the organisms in the fermentation medium. Ribosomal Database Project Classifier analysis identified 31 genera, among which Lactobacillus drastically increased during fermentation (150-fold increment over 8 d), while the relative populations of the other gram-positive bacteria (Staphylococcus and Bacillus) decreased. Basic Local Alignment Search Tool analysis revealed the dominant species to be L. sakei. This organism constituted approximately 90% of Lactobacillus and 79% of total microbiota. The taxonomic diversity and species richness (assayed by Shannon-Weiner Index and Chao 1, respectively) were not significantly different between middle-ages kaburazushi and ancient narezushi. Both types were characterized by the preferential growth of Lactobacillales.

Volatile compounds of headspace gas in the Japanese fish sauce ishiru

Volatile compounds from the headspace gas of ten brands of the Japanese fish sauce ishiru were analyzed by GC-MS with a thermal-desorption cold-trap system. Many volatile peaks were detected and 51 compounds were identified. The major volatile compounds in ishiru included aldehydes (such as 2-methylpropanal, 2-methylbutanal, 3-methylbutanal, and benzaldehyde), nitrogen-containing compounds (such as pyrazine derivatives and trimethylamine), sulfur-containing compounds (such as dimethyl disulfide), and ketones (such as 2-butanone and 3-methyl-2-butanone). On the other hand, volatile fatty acids were nearly absent in the headspace gas of ishiru.

Eucalyptus leaf extract inhibits intestinal fructose absorption, and suppresses adiposity due to dietary sucrose in rats

Sucrose is more lipogenic than starch, and the extreme ingestion of sucrose induces adiposity and obesity. The aim of this study was to examine the effect of the eucalyptus (Eucalyptus globulus) leaf extract (ELE) on adiposity due to dietary sucrose in rats. In addition, in this study, the effect of ELE on intestinal fructose absorption was also examined. Rats were fed a high-sucrose diet (75 % in calorie base) with or without ELE (10 g/kg diet) for 5 weeks. Body weight was lower in the rats receiving ELE than in the controls (342 (sd 37.9) v. 392 (sd 26.0) g (n 7); P<0.05). Furthermore, ELE resulted in decreases in the triacylglycerol concentrations in the plasma (1.44 (sd 0.448) v. 2.79 (sd 0.677) mmol/l (n 7); P<0.05) and liver (19.1 (sd 5.07) v. 44.1 (sd 16.28) micromol/g (n 7); P<0.05). In contrast, ELE did not show any significant effects in the rats fed a starch diet. When rats were orally given ELE 10 min before fructose administration, the intestinal fructose absorption, which was examined by measuring the elevated concentration of fructose in the portal vein at 30 min after the fructose administration, was significantly inhibited in a dose-dependent manner. Furthermore, in rats fed a high-fructose diet, the plasma and hepatic triacylglycerol concentrations were significantly decreased by ELE. These results indicate that ELE, which inhibits the intestinal fructose absorption, can suppress adiposity in rats that ingest large amounts of sucrose or fructose.

The occurrence of internal browning induced by high soil temperature treatment and its physiological function in Raphanus root

Abstract Internal browning (IB), consisting of a brown coloration at the central stele of the root of radish ( Raphanus sativus ), is a physiological disorder induced by high temperature conditions. In this study, different durations of high soil temperature treatment were compared on the basis of their influence on both the occurrence of IB and its related enzyme fluctuations. In affected roots, the degree of IB was positively connected with a variation in the enzyme activities of polyphenol biosynthesis and the ascorbate–glutathione cycle. Accordingly, the heat stress led to significant increases of glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), l -phenylalanine ammonia-lyase (PAL), as well as polyphenol oxidase (PPO) activities and IB formation, in the susceptible cultivar Fukutenka, but not in the resistant cultivar Minowase. Although no correlation was found between catalase (CAT) activity and IB occurrence, a pronounced increase in ascorbate peroxidase (APX) and decreases of dehydroascorbate reductase (DHAR) and glutathione reductase (GSHR) activities were detected in roots suffering from IB. These observations strongly suggest that detoxification of H 2 O 2 by polyphenol biosynthesis was operating, and that the pentose phosphate pathway was activated in order to supply the NADPH needed for its biosynthesis.

Purification and immunological properties of fructose 2,6-bisphosphate-sensitive pyrophosphate: d-fructose 6-phosphate 1-phosphotransferase from the protist Euglena gracilis

Abstract 1. 1. Fructose 2,6-bisphosphate-sensitive pyrophosphate: d -fructose 6-phosphate 1-phosphotransferase (PPi-PFK) has been purified to homogeneity from Euglena gracilis SM-ZK. 2. 2. The enzyme had an apparent mol. wt of 110,000 and consisted of a single protein. 3. 3. The pH optimum for the forward and the reverse reactions was 6.5–6.7. 4. 4. In the forward reaction, the saturation curve for Fru 6-P was sigmoidal and apparent K 0.5 value was 2.5 mM, while that for PPi was hyperbolic and K m value was 100 μM. 5. 5. In the presence of Fru 2,6-P2, the affinity for Fru 6-P was greatly increased; the kinetics were hyperbolic and K m value was 0.3 mM. The K a value for Fru 2,6-P2 was as low as 38 nM. 6. 6. In the reverse reaction, the saturation curves for both Fru 1,6-P2 and Pi were hyperbolic. 7. 7. In the presence of Fru 2,6-P2, the affinity for Pi was decreased. 8. 8. On double diffusion in agar gel, the antibody of PPi-PFK gave a single precipitin line against the purified Euglena enzymes, and it gave no precipitin line against PPi-PFK from other sources.

Regulation of glycolysis during acclimation of scallops (Patinopecten yessoensis Jay) to anaerobiosis.

Some glycolytic metabolites in the adductor muscle were measured after transfer of scallops from aerobic to anaerobic saltwater for 12 h. The level of octopine increased gradually during the initial 3 h incubation, and thereafter the level increased rapidly up to 12 h. The ATP level also did not show any significant change for the initial 3 h, and then decreased rapidly. The fructose 2,6-biphosphate (Fru 2,6-BP) level increased drastically during the initial 3 h incubation, but thereafter the level did not show any significant change up to 12 h. In the short-term effects of anaerobiosis for 90 min, the level of fructose 6-phosphate (Fru 6-P) increased just after transfer to anaerobiosis, and then its level decreased. In contrast, the fructose 1,6-biphosphate (Fru 1,6-BP) level increased greatly, at the time when both glucose 6-phosphate (Glc 6-P) and Fru 6-P decreased. The Fru 2,6-BP level did not any significant change during the initial 15 min incubation, but thereafter the level increased gradually up to 90 min. Scallop 6-phosphofructo 1-kinase (EC 2.7.1.11) (PFK1) was strongly activated by 1 microM Fru 2,6-BP when 0.2 mM Fru 6-P was used as a substrate, but the activity was not affected at 5 mM Fru 6-P. In view of these results, the regulation mechanism of glycolysis is discussed.

Hypoxia-induced megaloblastosis in vitamin B12-deficient rats

In rats, in contrast with human subjects who develop megaloblastic anaemia due to vitamin B12 deficiency, haematological abnormalities with anaemia were not observed under normoxic conditions even though plasma vitamin B12 concentration was reduced to <15 % of a normal concentration by depleting dietary vitamin B12. To elucidate whether erythropoiesis was affected by vitamin B12 deficiency in rats, these vitamin B12-deficient rats were exposed to hypoxia (10.5 % O2) to stimulate erythropoiesis. In the vitamin B12-sufficient control rats, erythrocyte count was significantly (P<0.05) increased 1 week after starting the hypoxic exposure. However, the hypoxia-induced erythropoiesis was affected by vitamin B12 deficiency, and no significant increase in the erythrocyte count was observed even after 6-week exposure to hypoxia in the vitamin B12-deficient rats. In the vitamin B12-deficient rats in hypoxia, erythrocytes became abnormally enlarged, and haemoglobin concentration in peripheral blood was increased in proportion to the increase of mean corpuscular volume. However, the level of the increase in the haemoglobin concentration was significantly (P<0.05) lower in the vitamin B12-deficient rats compared with that in the -sufficient controls. In addition, in the vitamin B12-deficient rats, in contrast to the -sufficient rats, serum erythropoietin concentration was not normalized even after 6-week exposure to hypoxia. These results indicate that a megaloblastic anaemia-like symptom is induced when the vitamin B12-deficient rats are exposed to hypoxia.