Masao Takeuchi

Theaflavin attenuates ischemia–reperfusion injury in a mouse fatty liver model

The incidence of non-alcoholic fatty liver disease (NAFLD) has been increasing, and there is a shortage of liver donors, which has led to the acceptance of steatotic livers for transplantation. However, steatotic livers are known to experience more severe acute ischemia-reperfusion (I/R) injury than normal livers upon transplantation. In the present study, we investigated the role of theaflavin, a polyphenol substance extracted from black tea, in attenuating acute I/R injury in a fatty liver model. We induced I/R in normal and steatotic livers treated with or without theaflavin. We also separated primary hepatocytes from the normal and steatotic livers, and applied RAW264.7 cells, a mouse macrophage cell line, that was pretreated with theaflavin. We observed that liver steatosis, oxidative stress, inflammation and hepatocyte apoptosis were increased in the steatotic liver compared to the normal liver, however, these changes were significantly decreased by theaflavin treatment. In addition, theaflavin significantly diminished the ROS production of steatotic hepatocytes and TNF-α production by LPS-stimulated RAW264.7 cells. We concluded that theaflavin has protective effects against I/R injury in fatty livers by anti-oxidant, anti-inflammatory, and anti-apoptotic mechanisms.

A sensitive method to screen for hydroxyl radical scavenging activity in natural food extracts using competitive inhibition ELISA for 8-hydroxy deoxyguanosine

Representative endogenous antioxidants and natural food extracts were screened for hydroxyl radical scavenging activity by an ELISA. Whereas conventional assays for hydroxyl radical scavenging activity use either spin traps following the induction of Fenton reaction or measure thiobarbituric acid-reactive substances, this assay measures 8-hydroxy deoxyguanosine liberated from the hydroxylation of deoxyguanosine by Cu2+/ascorbate system.

Determination of HEL (Hexanoyl-Lysine Adduct): A Novel Biomarker for Omega-6 PUFA Oxidation

Published evidences indicate that reactive oxygen species (ROS) can induce lipid peroxidation, which plays important role in the pathophysiology of numerous diseases including atherosclerosis, diabetes, cancer and aging process. Monitoring of oxidative modification or oxidative damages of biomolecules may therefore be essential for the understanding of aging, and age-related diseases. N-epsilon-Hexanoyl-lysine (HEL) is a novel lipid peroxidation biomarker which is derived from the oxidation of omega-6 unsaturated fatty acid. In this chapter, development of HEL ELISA and its applications are reported. Assay range of HEL ELISA was 2-700 nmol/L, and showed good linearity and reproducibility. Accuracy of this assay was validated by recovery test and absorption test. HEL concentration in human urine was 22.9 ± 15.4 nmol/L and it was suggested that HEL exists as low molecular substances, in a free or in the peptide-attached form. In contrast with the urine sample, serum HEL was suggested to exist in the protein-attached form, and hydrolysis by protease might be essential for the accurate measurement of HEL in protein containing samples such as serum and cultured cells. By sample pretreatment with proteases, HEL was successfully detected in oxidized LDL, oxidized serum, and rat serum. In conclusion, HEL ELISA can be applied to measure urine, serum, and other biological samples independent of the animal species, and may be useful for the assessment of omega-6 PUFA oxidation in the living bodies.

Superoxide Radical Scavenging Activities of Wines, and Antioxidative Properties of Fractions Recovered from Merlot Wine Pomace

Wine samples (n = 43) differing in origin, grape variety, and vintage were analyzed for total phenolic content, sulfur dioxide content, color (OD520), and superoxide radical scavenging activities (SOSA). A direct correlation between the wine color (r = 0.7517), its phenolic content (r = 0.9686), and the ability of the wine constituents to scavenge superoxide radicals was established using a simple regression analysis. Distribution of the SOSA in wines was further examined by fractionating 12 wines into three fractions with a C18 Sep-pak cartridge. The correlation coefficient between SOSA and total phenol content in the fractions was r = 0.2705 in fraction A (containing phenolic acids, sugars, organic acids, amino acids, and salts) r = 0.9575 in fraction B (containing procyanidins, flavonols, and catechin) and r = 0.9785 in fraction C (containing anthocyanins and tannins), respectively. As the phenolic content of fraction C was about twofold higher than that of fraction B, fraction C contributed most to the SOSA value in wine. Phenolic compounds were extracted with 50% ethanol from Merlot wine pomace. The extract showed high antioxidant activities, and also scavenged the superoxide and hy-droxyl radicals quite efficiently.

Antioxidative Defense System and Free Radical Scavenging Potentials of Cereals

Cereals occupy an important place in the human diet. In addition to being the primary source of carbohydrates, cereals also provide us with vitamins, trace minerals, and dietary fibers. With the recent increase in interest in the intake of dietary antioxidants, cereals have also been investigated for their antioxidant defense potentials. This review focuses on the description of a phenolic antioxidant defense system in the rice hull that has been demonstrated to offer chemical protection to the rice grain from undergoing oxidative damage. In addition, data are presented to demonstrate the free radical scavenging potentials of the hot aqueous extracts of some of the selected cereal grains. Information is also provided on the newly identified antioxidants in wild rice, and in the green leaves of young barley plants.

Effect of L-Carnosine on 8-OH Deoxyguanosine Release by H2O2 in 3Y1 Rat Embryo Fibroblasts

L-Carnosine is abundantly present in vertebrate skeletal muscles. Its antioxidant role in the body has been of interest to nutritional biochemists. We investigated the release of 8-hydroxydeoxyguanosine (8-OH dG), one of the markers for in vivo oxidative DNA damage, by rat embryo fibroblasts in response to oxidative stressor H2O2 and L-carnosine. When fibroblasts were exposed only to H2O2 (10–500 μM) for 120 minutes varying patterns of 8-OH dG release were observed with increasing concentration of H2O2. At a concentration of 100 μM, H2O2 produced a dose-dependent increase in 8-OH dG release into the medium. Exposure of fibroblasts to 500 μM of H2O2, even for 30 minutes, resulted in irreversible cell death. The antioxidant effect of L-carnosine was measured by exposing the fibroblasts simultaneously to H2O2 (250 μM) and L-carnosine (10 or 30 mM) for 60 minutes. The 8-OH dG release from the fibroblasts treated with H2O2 and L-carnosine was markedly lower than that of fibroblasts treated with H2O2 only. We conclude that the antioxidant role of L-carnosine is probably executed via its inhibition of the formation of 8-OH dG.