[Identification of Plant Fragments by Analyzing the Plastid rpl16-rpl14 Linker Sequences].

An identification method for testing contamination in products was assessed using various vegetables and fruits (70 types in total). DNA was extracted from plant fragments which are 1 to several millimeters long and the plastid rpl16-rpl14 linker sequence (approximately 550 base pairs) was amplified by PCR. The DNA nucleotide sequence was determined, and homology and SNP (single nucleotide polymorphism) analyses were carried out. Consequently, the test plants were difficult to distinguish between closely related species, but could be divided into 38 groups at the genus level or the species level. Although problems such as the accuracy of discrimination among some closely related plants and DNA stability under an acidic condition remain to be resolved, this method is considered to be expected to identify plant fragments mixed in products or raw materials.

Differentiation of Laboratory Strains of Red Flour Beetle Tribolium castaneum Collected from Various Areas.

We recently reported polymorphism in the mitochondrial genes of Tribolium castaneum, a stored grain pest. Here, we show that T. castaneum samples from the main island of Japan can be easily differentiated from samples from other areas by using real-time PCR with minor groove binding (MGB) TaqMan probes containing a single nucleotide polymorphism (SNP) to amplify a 179-bp fragment. This assay was also applicable to samples stored at room temperature for 5 months after natural death, and samples whose DNA was artificially degraded by heat, pressure, or gamma-ray irradiation.

Polymorphism observed in mitochondrial genes of red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae) of different origin in laboratory cultures.

Tribolium castaneum, a pest of stored grain, has a uniform morphology, preventing the visual identification of strains from different areas. Polymorphisms in the nucleotide sequences of the mitochondrial genes of this species were examined, and combined into seven haplotypes among the test insect specimens originating from Japan, Thailand, and Canada. These results suggested the potential for geographical differentiation.

[Determination of Insect Order by Analyzing Mitochondrial Gene ND5].

Polymorphism in the mitochondrial gene ND5 enables the determination of the order, a taxonomic rank, of many insects from the 5 major insect orders-Diptera, Hymenoptera, Hemiptera, Coleoptera, and Lepidoptera. DNA nucleotide sequences of multiple species were determined and more than 19 nucleotides characteristic of each order were identified; this makes it possible to discriminate insects at the order level more precisely and easily than when using conventional techniques.

[Structural analysis of subsidiary tar dyes contained in Food red No. 102].

Three unknown colors (A1, A2 and A3) in Food red No. 102 (R102) were identified by LC-MS and NMR analysis. Red dye A1 was azo rubine (AZO), which is not designated as a coloring agent in Japan. Red dye A2 was a structural isomer of Fast Red E (FRE), which is also not designated as a coloring agent in Japan. Red dye A3 was predicted to be an overreaction product of R102, possessing the R102 structural framework. Furthermore, AZO, which was assumed to have been produced by dimerization of a diazonium salt used as a synthetic intermediate of R102, was also identified in Food red No. 2 (R2).

Evaluation of antioxidative effects of sesamin on the in vivo hepatic reducing abilities by a radiofrequency ESR method.

Antioxidative effects of sesamin (a mixture of sesamin and episesamin) were evaluated in the liver, kidney and inferior vena cava of living rats using a radiofrequency ESR method. TEMPOL, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl, was used as an in vivo redox probe, the half-life of which is believed to be correlated with the antioxidant status. The oral administration of sesamin (250 mg/kg rat weight) 3 h before ESR measurements shortened the half-life of TEMPOL in the liver by 10 - 15% as compared with the controls, but did not affect the other organs. This effect was maintained for at least 3 h after the administration, and then disappeared at 24 h, corresponding to the results of our preliminary pharmacokinetic studies. Changes in the reducing ability were observed only in the hepatic sites of the sesamin-treated rats. These findings suggest that sesamin exhibits effective antioxidant activity in the liver via modulation of the intracellular redox status related to TEMPOL reduction.

Novel antioxidative metabolites in rat liver with ingested sesamin.

Sesamin, a major lignan in sesame oil, is known to have many biological activities, especially protective effects against oxidative damage in the liver. As sesamin itself has no antioxidative properties in vitro, to elucidate the mechanism of its antioxidative effects, the reaction products of sesamin in rat liver homogenate were analyzed. The methylenedioxyphenyl moiety in the structure of sesamin was shown to be changed into a dihydrophenyl (catechol) moiety. The enzymatic reaction products in vitro were identified as (1R,2S,5R,6S)-6-(3,4-dihydroxyphenyl)-2-(3,4-methylenedioxyphenyl)-3,7-dioxabicyclo[3,3,0]octane and (1R,2S,5R,6S)-2,6-bis(3,4-dihydroxyphenyl)-3,7-dioxabicyclo[3,3,0]octane, which showed strong radical scavenging activities; the latter was a novel compound. The same metabolites were found as glucuronic acid and/or sulfic acid conjugates in substantial amounts in rat bile after oral administration of sesamin. It is suggested that sesamin is a prodrug and the metabolites containing the catechol moieties in their structures are responsible for the protective effects of sesamin against oxidative damage in the liver.

Absorption and metabolism of antioxidative polyphenolic compounds in red wine.

We have shown that drinking red wine reduces oxidation of LDL. This reduction in oxidation has been attributed to the polyphenolic compounds in red wine, but the mechanisms of absorption and metabolism of these compounds has been unclear. We therefore investigated the absorption and metabolism of polyphenols using rats to identify their active forms in biological fluids. We also investigated the effect of tartaric acid (TA), a major organic acid in wine, on the absorption of polyphenols. Our results suggested that low molecular weight polyphenols are absorbed in the intestine and metabolized to their glucuronide conjugates, which exhibit antioxidative activity in plasma, and that TA can enhance the bioavailability of wine polyphenols.